Somatostatin analogs with inhibitory activity to growth hormone release

ABSTRACT

Provided are therapeutic and diagnostic somatostatin analogs including radiotherapeutic and radiodiagnostic reagents, and methods of making and use thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional and claims priority to U.S. patentapplication Ser. No. 10/568,112, filed Sep. 27, 2006 now U.S. Pat. No.7,968,080, which is a U.S. National Stage application filed under 35U.S.C. 371 and claims priority to PCT/US04/27128, filed Aug. 20, 2004,which claims priority under 35 U.S.C. §119 to provisional applicationSer. No. 60/496,942, filed Aug. 20, 2003, the disclosures of which areincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support of Grant No. DK 15410,awarded by the National Institutes of Health. The Government has certainrights in this invention.

TECHNICAL FIELD

This invention relates to somatostatin analogs and more particularly tosomatostatin analogs that specifically interact with certainsomatostatin receptor subtypes.

BACKGROUND

Somatostatin (SS) is an endogenous peptide that acts as a hormone,neurotransmitter and neuromodulator, as well as a paracrine regulator ofneighboring cells. It is present in two forms, SS-14 and SS-28, atetradecapeptide and a 28 amino acid peptide, which originate in theprotein preprosomatostatin and are distributed differently inneuroendocrine and central and peripheral nervous system tissues. Thereare five subtypes of somatostatin receptors, all G protein-coupledreceptors with a high sequence homology.

Despite the commercial availability of octreotide, lanreotide, andvapreotide, a large number of somatostatin analogs have been proposedfor use as imaging and/or therapeutic agents to detect and/or treatcancer and other somatostatin-responsive disease states. Analogs withhigher affinity to somatostatin receptors (SSTs) and to SST subtypes, inparticular to SST2 and SST5 are desirable, such that lower dosages ofsomatostatin analogs may be administered to obtain a clinical response.

SUMMARY

The invention provides cyclic peptidomimetic somatostatin analogs, whichare designed to satisfy the need for potent and selective SST2 and SST5ligands. The presence of the functionalized aromatic amino acids opensthe way to new “handles” for additional functionalization and broaderapplications.

The analogs of the invention are beneficial anti-tumor agents. They maybe used for the treatment of acromegaly and diabetes, as well as forscintigraphy purposes when radioactively labeled. In addition, theanalogs may be radioactively labeled and/or linked to cytotoxic agentscapable of causing cell death (e.g., tumor cell death).

The compositions (i.e., the SS analogs) of the invention are useful asligands that are designed to interact with certain SST subtypes (e.g.,SST2 and SST5) on cells in vitro and in vivo.

The invention provides a somatostatin (SS) analog, wherein the analog isselected from any one of4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂;4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂;4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Asp-NH₂;4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Thr-NH₂;4-amino-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂;4-amino-3-iodo-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂;4-amino-3-iodo-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂;4-amino-3-iodo-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂;4-amino-3-iodo-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂; andD-Phe-C[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂. In one aspect of theinvention, the SS analog can be linked to a radioactive element.

The invention further provides methods of visualizing malignant cells ina subject comprising administering to the subject an SS analog of theinvention.

Also provided by the invention is a method of treating variousproliferative disorders in a subject comprising administering to thesubject an SS analog of the invention. In one aspect of the inventionthe proliferative disorder comprises a tumor, acromegaly, and/ordiabetes.

The invention also provides somatostatin (SS) analogs that are designedto bind selectively to SS receptor 2 (SST2) and/or SST5 in contrast toother SS receptors, wherein the SS analog has a structure selected fromthe group consisting of4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂;4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂;4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Asp-NH₂;4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Thr-NH₂;4-amino-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂;4-amino-3-iodo-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂;4-amino-3-iodo-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂;4-amino-3-iodo-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂;4-amino-3-iodo-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂; andD-Phe-C[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ and compounds containing di-or polyiodinated aromatic residues.

The invention also provides a pharmaceutical composition comprising amixture of an SS analog of the invention and at least onepharmaceutically acceptable carrier.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

DETAILED DESCRIPTION

Somatostatin inhibits the release of insulin and glucagon from thepancreas, inhibits growth hormone release from the pituitary and reducesgastric secretions. The half-life in plasma of native somatostatin isless than 3 minutes. It is rapidly degraded by peptidases. As aconsequence, somatostatin analogs with improved bioavailability, as wellas receptor specificity, are currently being sought. Somatostatin andits analogs are likely to be involved with treatment of variousdiseases. The number and variety of diagnostic and therapeutic uses forSS analogs, especially for receptor-specific peptidomimetic andnon-peptidic receptor-specific ligands have proliferated.

Numerous tissues in the human body express somatostatin receptorsincluding, but not limited to: (1) the gastrointestinal tract, (2) theperipheral nervous system, (3) the endocrine system, (4) the vascularsystem, and (5) lymphoid tissue, where the receptors are located ingerminal centers. In all these cases, somatostatin binding is of highaffinity and specific for bioactive somatostatin analogs. After bindingof ligands to somatostatin receptors, the agonist-receptor complexes areinternalized by cells. This property is important practically, andconstitutes the basis of localization and treatment of tumors whichover-express somatostatin receptors.

Somatostatin receptors are also expressed in pathological states,particularly in neuroendocrine tumors of the gastrointestinal tract.Most human tumors originating from the somatostatin target tissue haveconserved their somatostatin receptors. It was first observed in growthhormone-producing adenomas and TSH-producing adenomas; about one-half ofendocrine inactive adenomas display somatostatin receptors. Ninetypercent of the carcinoids and a majority of islet-cell carcinomas,including their metastasis, usually have a high density of somatostatinreceptors. However, only 10 percent of colorectal carcinomas and none ofthe exocrine pancreatic carcinomas contain somatostatin receptors. Thesomatostatin receptors in tumors can be identified using in vitrobinding methods or using in vivo imaging techniques; the latter allowthe precise localization of the tumors and their metastases in subjects.Because somatostatin receptors in gastroenteropancreatic tumors arefunctional, their identification can be used to assess the therapeuticefficacy of an analog to inhibit excessive hormone release subjects.

The cyclic tetradecapeptide somatostatin-14 (SS-14) was originallyisolated from the hypothalamus and characterized as an inhibitor ofgrowth hormone release from the anterior pituitary (see, e.g., U.S. Pat.No. 3,904,594, incorporated herein by reference). This tetradecapeptidehas a bridging or cyclizing bond between the sulfhydryl groups of thetwo cysteinyl amino acid residues in the 3 and 14 positions. SS-14regulates insulin, glucagon, and amylase secretion from the pancreas,and gastric acid release in the stomach. For example, SS-14 inhibits theeffects of pentagastrin and histamine on the gastric mucosa. SS-14 isalso expressed in intrahypothalamic regions of the brain and has a rolein the regulation of locomotor activity and cognitive functions. SS-14is present throughout the central nervous system and acts as aneurotransmitter. In the central nervous system, SS-14 has been shown toboth positively and negatively regulate neuronal firing, to affect therelease of other neurotransmitters, and to modulate motor activity andcognitive processes.

SS-14 affects multiple cellular processes. Studies have shown that SS-14is an inhibitory regulator of adenylyl cyclase in different tissues.SS-14 also regulates the conductance of ionic channels, including bothpotassium and calcium channels. These actions of SS-14 are mediated viapertussis toxin-sensitive guanine nucleotide-binding proteins. SS-14also regulates the activity of tyrosine phosphatases and cellularproliferation through pertussis toxin-insensitive mechanisms.

SS-14 induces its biological effects by interacting with a family ofmembrane-bound structurally similar receptors. Five SS-14 receptors havebeen cloned and are referred to as SST 1-5. Human SST1, mouse SST2 andmouse SST3 are described in Raynor et al., Molecular Pharmacology, 43,838-844 (1993), and all five human SS receptors are now available forresearch purposes. Human SST1, 2 and 3 are also disclosed in U.S. Pat.No. 5,436,155. Additional SS-14 receptors are disclosed in U.S. Pat.Nos. 5,668,006 and 5,929,209. All five receptors bind SS-14 and SS-28with high affinity. Selective agonists of SST2 and SST5 have beenidentified and used to reveal distinct functions of these receptors.These two receptors are believed to be the predominant subtypes inperipheral tissues. SST2 is believed to mediate the inhibition of growthhormone, glucagon and gastric acid secretion. In contrast, SST5 appearsto be primarily involved in the control of insulin and amylase release.SST3 is found in cortex tissue, in the pituitary and in adenoma tumortissue; it is believed to mediate inhibition of gastric smooth musclecontraction upon binding by SS-14. These findings indicate thatdifferent receptor subtypes mediate distinct functions of SS-14 in thebody.

Somatostatin binds to five distinct receptor (SSTs) subtypes withrelatively high affinity for each subtype. Binding of agonists to thedifferent SST subtypes have been associated with the treatment of thefollowing conditions and/or diseases. Activation of types 2 and 5 havebeen associated with growth hormone suppression and more particularly GHsecreting adenomas (Acromegaly) and TSH secreting adenomas. Activationof type 2 but not type 5 has been associated with treatingprolactin-secreting adenomas. Other indications associated withactivation of the somatostatin subtypes are restenosis, inhibition ofinsulin and/or glucagon and more particularly diabetes mellitus,hyperlipidemia, insulin insensitivity, Syndrome X, angiopathy,proliferative retinopathy, dawn phenomenon and Nephropathy; inhibitionof gastric acid secretion and more particularly peptic ulcers,enterocutaneous and pancreaticocutanieous fistula, irritable bowelsyndrome, Dumping syndrome, watery diarrhea syndrome, AIDS-relateddiarrhea, chemotherapy-induced diarrhea, acute or chronic pancreatitisand gastrointestinal hormone secreting tumors; treatment of cancer suchas hepatoma; inhibition of angiogenesis, treatment of inflammatorydisorders such as arthritis; chronic allograft rejection; angioplasty;preventing graft vessel and gastrointestinal bleeding. Somatostatinagonists can also be used for decreasing body weight in a patient.

Somatostatin-28 (SS-28) was isolated from porcine upper small intestine(L. Pradayrol, et al. in FEBS Letters 109:55-58, 1980). SS-28 is anN-terminally extended version of SS-14 that has an additional 14 aminoacid residues and which shows some increased potency when administeredin vivo.

A cyclic SS-14 analog, termed SMS-201-995 (Octreotide), i.e.D-Phe-c[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-ol is being used clinically toinhibit certain tumor growth. This analog has been shown to improvequality of life for the treated subject and there is strong evidence forcontrol of tumor growth and reduction in mortality. Two similaroctapeptide analogs, i.e. Lanreotide(D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂) and Vapreotide(D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Trp-NH₂), have also been developed,see Smith-Jones et al., Endocrinology, 140, 5136-5148 (1999). Thesesomatostatin analogs have been developed for use in radioimaging or asradiopharmaceuticals in radionuclide therapy. For radioimaging labelingwith ¹²³I can be used as disclosed in U.K. Patent Application 8927255.3and as described in Bakker et al., J. Nucl. Med., 32:1184-1189, 1991.Typically proteins have been radiolabeled through the use of chelatingagents, and there are various examples of complexing somatostatinanalogs with ⁹⁹Tc, ⁹⁰Y or ¹¹¹In, see U.S. Pat. Nos. 5,620,675 and5,716,596. For example Octreotide scintigraphy is based on thevisualization of octreotide-binding receptor(s). For these purposes, aradiolabeled form of octreotide, such as [¹²³I-Tyr³]-octreotide wasused. This and other developed analogs (Lanreotide(D-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2; VapreotideHD-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Trp-NH₂); AN-238, which is RC-121(D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂) linked to a cytotoxicagent), are part of the arsenal presently available in the anti-cancerarena.

Somatostatin agonists have also been disclosed to be useful forinhibiting the proliferation of Helicobacter pylori.

Octreotide and other clinically used SS-14 analogs interactsignificantly with three of the receptor subtypes, i.e. SST2, SST3 andSST5. SST2 and SST5 have been reported to mediate antiproliferativeeffects of SS-14 on tumor cell growth; therefore, they may mediate theclinical effects of Octreotide in humans. For example, compound RC-121is less potent than natural SS-14 and sandostatin, but presents theadvantage of binding selectively to receptors SST2 and SST5 (see, e.g.,Table 1).

TABLE 1 Binding Affinities of SS-14, Sandostatin, and RC-121 K_(i)(nM) ±SEM Peptide SST1 SST2 SST3 SST4 SST5 SS-14  2.3 ± 0.47 0.23 ± 0.04 1.17± 0.23 1.7 ± 0.3 1.4 ± 0.3 Sandostatin 875 ± 180 0.57 ± 0.08 26.8 ±7.7  >1000 6.8 ± 1.0 RC-121 >1000 1.7 ± 0.5 >1000 >1000 13.1 ± 1.2 

The invention provides methods and compositions that selectivelyinteract with SST2 and/or SST5. It is believed that the inhibition ofgrowth hormone release is mediated through interaction of ligands withthe SST2 receptor, or that both SST2 and SST5 are implicated. It is alsobelieved that interaction with SST5 is responsible for the insulinrelease inhibitory activity. Thus, analogs that are selective to eitheror both of these receptors would be useful for the treatment of cancerand diabetes among other uses.

The invention provides SS analogs having a modified terminal residue(s)with acidic and basic residues and/or peptidomimetic building blocks toimprove potency, selectivity and bioavailability. The SS-14 analogs ofthe invention were developed based upon the parent compound (RC-121D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂, because of the bindingprofile of this parent compound. The parent compound is selective forSST2 and SST5 (see, e.g., Table 1), but its properties can be improvedfrom the standpoint of potency, stability in the biodomain and ratio ofpotencies at SST2 and SST5.

The SS-14 analogs of the invention provide several attractive featuresincluding (1) the analogs are readily synthesized from commerciallyavailable building blocks; (2) peptidomimetic modifications, designed toincrease bioavailability (e.g., p-amino-D-phenylalanine,(2-amino-3-(4-aminophenyl)-propanoic acid) and 3-iodo-tyrosine,(2-amino-3-(4-hydroxy-3-iodophenyl)-propanoic acid)); (3) potent andselective-compound(4-amino-3-iodo)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂, which isdesigned to inhibit growth hormone in vitro more effectively thanoctreotide, used as a reference compound herein; and (4) the analogs arebased, in part, on iodoaryl derivatives, such that radioactive labelingof these compounds is readily achieved using radioactive iodine. Suchmolecules are likely to have applications in visualization anderadication of malignant cells.

The invention provides SS-14 analogs comprising disulfide-bridgedoctapeptides incorporating non-natural amino acid building blocks. Theinvention provides SS-14 analogs of general formula I:X-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Y—NH₂, wherein X is selected from thegroup consisting of D-Phe, (4-amino)-D-Phe, and (4-amino-3-iodo)-D-Phe,wherein Y is selected from the group consisting of L- or D-Thr and L- orD-Asp, and wherein the Tyr at position 3 can be mono- or polyiodinated.Exemplary peptide structures according to formula I are provided inTable 2.

TABLE 2 No. Compound 1 D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ 2(4-amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ 3(4-amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ 4(4-amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Thr-NH₂ 5(4-amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Asp-NH₂ 6(4-amino)-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr- NH₂ 7(4-amino-3-iodo)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ 8(4-amino-3-iodo)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ 9(4-amino-3-iodo)-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]- Thr-NH₂ 10(4-amino-3-iodo)-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]- Asp-NH₂

The nomenclature used to define the peptides is as described in MGoodman, A Felix, L Moroder and C Toniolo (Eds.). Synthesis of Peptidesand Peptidomimetics, (2003) wherein, in accordance with conventionalrepresentation, the amino group appears to the left and the carboxylgroup to the right. The standard 3-letter abbreviations to identify thealpha-amino acid residues, and where the amino acid residue has isomericforms, it is the L-form of the amino acid that is represented unlessotherwise expressly indicated, e.g. Ser=L-serine. By D,L is meant amixture of the D- and L-isomers of a particular α-amino acid.

In another aspect, the invention provides a pharmaceutical compositioncomprising an effective amount of a compound of formula (I) or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier.

In still another aspect, the invention provides a method of eliciting asomatostatin receptor agonist effect in a subject in need thereof, whichcomprises administering to said mammal an effective amount of a compoundof formula (I) or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a method of treatingprolactin-secreting adenomas, restenosis, diabetes mellitus,hyperlipidemia, insulin insensitivity, Syndrome X, angiopathy,proliferative retinopathy, dawn phenomenon, Nephropathy, gastric acidsecretion, peptic ulcers, enterocutaneous and pancreaticocutaneousfistula, irritable bowel syndrome, Dumping syndrome, watery diarrheasyndrome, AIDS-related diarrhea, chemotherapy-induced diarrhea, acute orchronic pancreatitis, gastrointestinal hormone secreting tumors, cancer,hepatoma, angiogenesis, inflammatory disorders, arthritis, chronicallograft rejection, angioplasty, graft vessel bleeding orgastrointestinal bleeding, in a subject in need thereof, which comprisesadministering to the subject a compound of formula (I) or apharmaceutically acceptable salt thereof.

In another aspect, this invention provides a method of inhibiting theproliferation of Helicobacter pylori in a subject in need thereof, whichcomprises administering to the subject a compound of formula (I) or apharmaceutically acceptable salt thereof.

A therapeutically effective amount of a peptide of the invention and apharmaceutically acceptable carrier substance together form atherapeutic composition (e.g., a pill, tablet, capsule, or liquid) foradministration (e.g., orally, intravenously, intravitreally,transdermally, pulmonarily, vaginally, subcutaneously, nasally,iontophoretically, or by intratracheally) to a subject in need of thepeptide. The pill, tablet, or capsule can be coated with a substancecapable of protecting the composition from the gastric acid orintestinal enzymes in the subject's stomach for a period of timesufficient to allow the composition to pass undigested into thesubject's small intestine. The therapeutic composition can also be inthe form of a biodegradable or nonbiodegradable sustained releaseformulation for subcutaneous or intramuscular administration. Continuousadministration can also be obtained using an implantable or externalpump to administer the therapeutic composition.

The dose of a peptide or therapeutic composition of the invention fortreating the above-mentioned diseases or disorders varies depending uponthe manner of administration, the age and the body weight of thesubject, and the condition of the subject to be treated, and ultimatelywill be decided by the attending physician or veterinarian. Such anamount of the peptide or therapeutic composition as determined by theattending physician or veterinarian is referred to herein as a“therapeutically effective amount.”

Selectivity for binding of the analog peptides of the invention to SST2and/or SST5 can be demonstrated by testing their interaction with thefive different cloned human SS-14 receptors. In vitro assays for theability of analogs to bind to various somatostatin receptor subtypes aredescribed herein and are known in the art (see, e.g., U.S. Pat. No.6,602,849; and U.S. Pat. No. 6,001,801, the disclosures of which areincorporated herein by reference). Generally, recombinant cellsexpressing the receptor are washed and homogenized to prepare a crudeprotein homogenate in a suitable buffer, as known in the art. In atypical assay, an amount of protein from the cell homogenate is placedinto a small volume of an appropriate assay buffer at an appropriate pH.Candidate substances, such as the analogs of the invention, are added tothe admixture in convenient concentrations, and the interaction betweenthe candidate substance (e.g., the analogs of the invention) and thereceptor polypeptide is monitored. The peptides of the invention aredesigned to bind substantially to certain SST subtypes such as SST2and/or SST5 with high affinity.

Receptor binding assays can be performed on cloned SS-14 receptors.Using such assays, one can generate K_(D) values which are indicative ofthe concentration of a ligand necessary to occupy one-half (50%) of thebinding sites on a selected amount of a receptor or the like, oralternatively, competitive assays can generate IC₅₀ values which areindicative of the concentration of a competitive ligand necessary todisplace a saturation concentration of a target ligand being measuredfrom 50% of binding sites.

Although an analog of octreotide has been employed to detect humantumors having high expression of SS-14 receptors through the use ofpositron-emission tomography, the existing SS-14 analogs do notdistinguish among SST2, SST3 and SST5. In comparison, radiolabeled SS-14analogs of the invention can be employed for similar purposes, and theyare considered to be specifically useful in identifying tumorsexpressing SST2 and/or SST5, which tumors are then therapeutic targetsfor treatment with the selective analogs of the invention labeled withcytotoxic and/or radioactive agents.

The SS-14 analogs of the invention are designed to selectively interactwith SST2 and/or SST5 and are useful in combating cancers, which expressSST2 and/or SST5. They are also useful in scintigraphy to determine thedistribution of cells and tissues expressing SST2 and/or SST5 receptorin the brain and in the endocrine and exocrine systems, and also inidentifying selective functions of this receptor in the body. They arefurther useful for treating non-neoplastic disorders linked to SST2and/or SST5-expressing tissues.

The invention also includes a combination of the peptide analogs of theinvention and a cytotoxic drug, such as paclitaxel or any othercytotoxic moiety. The cytotoxic drug can be linked to the analog througha covalent bond or a physical encapsulation.

In general, an SS-14 analog is synthesized on a solid-phase peptidesynthesizer using Fmoc chemistry. The desired compound, such asanticancer drugs paclitaxel, doxorubicin or camptothecin or the like,that is intended to be delivered to the target cells, reacts with aspacer (typically having a carboxyl terminal group) to form a covalentbond, resulting in a drug-spacer complex. Such complex is then coupledto the N-terminal of the SS-14 analog peptide on the resin to form thefinal product, namely, a drug-spacer-peptide complex.

The compounds of the invention (e.g., peptide analogs) may be used inradiolabeled or unlabelled form to diagnose or treat anysomatostatin-responsive disease state. The compounds of the inventionare particularly useful for diagnosis and/or treatment of neoplasticdisorder and tumors such as, for example, neuroendocrine tumors,pituitary adenomas, pheochromocytomas, paragangliomas, medullary thyroidcarcinomas, small cell and non small cell lung cancers, astrocytomas,melanomas, meningiomas, breast tumors, malignant lymphomas, renal cellcarcinomas, prostate tumors, and the like. The SS-14 analogs of theinvention may also be used to diagnose or to treat conditions in whichangiogenesis and concomitant up-regulation of SSTs occurs. Suchconditions include, for example, atherosclerosis and cellularproliferation occurring in arteries after invasive procedures such asangioplasty.

Radiolabeled SS-14 analogs of the invention are useful for suchdiagnoses and treatments. Radiolabeled embodiments of the analogs of theinvention may be used in radioisotope guided surgery, as described in WO93/18797 and in Woltering, et al. (1994) Surgery 116, 1139-1147. In oneembodiment, a complex of a γ-emitting radionuclide and an analog of theinvention is used to diagnose an SST-expressing tumor, and subsequently,a complex of β-emitting radionuclide such as ¹⁸⁸Re or ¹⁸⁶Re with theanalog of the invention is used to treat the tumor. Other therapeuticradionuclide labels include such cytotoxic radioisotopes as scandium-47,copper-67, gallium-72, yttrium-90, iodine-125, iodine-131, samarium-153,gadolinium-159, dysprosium-165, holmium-166, ytterbium-175,lutetium-177, rhenium-186, rhenium-188, astatine-211 and bismuth-212.

For diagnostic purposes, an effective diagnostic amount of theradiolabeled analog of the invention is administered, typicallyintravenously. An effective diagnostic amount is defined as the amountof radiolabeled analog necessary to effect localization and detection ofthe label in vivo using conventional methodologies such as magneticresonance, computerized tomography, gamma scintigraphy, SPECT, PET, andthe like.

For diagnosis using scintigraphic imaging radiolabeled analogs of theinvention are typically administered in a single-unit injectable dose.The labeled analog provided by the invention may be administeredintravenously in any conventional medium for intravenous injection suchas an aqueous saline medium, or in blood plasma medium. Generally, theunit dose to be administered has a radioactivity of about 0.01 mCi toabout 100 mCi, typically 1 mCi to 50 mCi. The solution to be injected atunit dosage is from about 0.01 mL to about 10 mL. After intravenousadministration, imaging in vivo can take place in a matter of a fewminutes. However, imaging can take place, if desired, hours or evenlonger after the radiolabeled compound is injected into a subject. Inmost instances, a sufficient amount of the administered dose willaccumulate in the area to be imaged within about 0.1 of an hour topermit the taking of scintiphotos. Any conventional method ofscintigraphic imaging for diagnostic purposes can be utilized inaccordance with this invention.

When the radiolabeled compounds of the invention are used fortherapeutic purposes, they are radiolabeled with a therapeuticallyeffective amount of a cytotoxic radioisotope, typically ¹⁸⁸Re. Inaccordance with the invention, a therapeutically effective amount of acytotoxic radioisotope means the total amount of each active componentof the pharmaceutical composition or method that is sufficient to show ameaningful benefit to the subject, e.g., a reduction in the incidence orseverity of symptoms attributed to the somatostatin-responsive diseasestate, as compared to that expected for a comparable group of subjectsnot receiving the radiotherapeutic agent of the invention. When appliedto an individual active ingredient administered alone, the term refersto that ingredient alone. When applied to a combination, the term refersto combined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially, orsimultaneously. For the purposes of this invention, radiotherapyencompasses any therapeutic effect ranging from pain palliation to tumorablation or remission of symptoms associated with the particularsomatostatin-responsive disease being treated.

When used for radiotherapy, a complex of an SS-14 analog of theinvention and a cytotoxic radioisotope is administered to a subject,typically a mammal, including a human, in need of treatment for asomatostatin-responsive disease or disorder. In the radiotherapeuticmethod of the invention, an amount of cytotoxic radioisotope from about5 mCi to about 200 mCi may be administered via any suitable clinicalroute, typically by intravenous injection or by intratumoral injection.The radiotherapeutic complex of the invention may optionally beadministered in combination with a chemotherapeutic drug such astamoxifen, cisplatin, taxol, anti-angiogenic compounds, and the like.

When unlabeled compound is used for therapy of a somatostatin-responsivedisease or disorder, administration of the SS-14 analog is typicallyparenteral, and more commonly intravenous. The amount of unlabeled SS-14analog administered for therapy of a somatostatin-responsive disease ordisorder will depend upon the nature and severity of the condition beingtreated, and upon the nature of prior treatments which the subject hasundergone. Ultimately, the attending physician will decide the amount ofSS-14 analog with which to treat each individual subject. Initially, theattending physician will administer low doses of the SS-14 analog andobserve the subject's response. Larger doses of the SS-14 analog may beadministered until the optimal therapeutic effect is obtained for thesubject, and at that point the dosage is not increased further. It iscontemplated that the dosage of unlabelled SS-14 analog administered inthe therapeutic method of the invention should be in the range of about0.1 μg to about 100 μg compound per kg body weight. More commonly, thedosage of unlabelled SS-14 analog administered in the therapeutic methodof the invention is in the range of about 0.1 μg to about 100 μg SS-14analog per kg body weight. The unlabelled SS-14 analog of the inventionmay also optionally be administered in combination with achemotherapeutic drug.

The duration of therapy, whether with a radiopharmaceutical comprisingan SS-14 analog of the invention or with an unlabelled SS-14 analog ofthe invention, will vary, depending on the severity of the disease beingtreated and the condition and idiosyncratic response of each individualsubject. It is contemplated that the duration of each administration ofthe radiopharmaceutical of the invention will be in the range of aboutone to about 120 minutes of continuous intravenous administration. It iscontemplated that the duration of each administration of the unlabelledSS-14 analog of the invention will be in the range of about one to about120 minutes of continuous intravenous administration. Ultimately theattending physician will decide on the appropriate duration ofintravenous therapy using the labeled or unlabeled compounds of theinvention, whether administered alone or in combination with otherdrugs.

According to one aspect of the invention, a formulation comprising oneor more SS-14 analogs of the invention are provided along with apharmaceutically acceptable carrier. The formulation provides an activedose in the range of from about 10 μg/kg body weight to about 60 μg/kgbody weight of an SS-14 analog of the invention; typically about 10μg/kg to about 20 μg/kg.

The expression “pharmaceutically acceptable” is meant to includeingredients that are compatible with an SS-14 analog of the invention aswell as physiologically acceptable to a subject receiving the formation,e.g. a human, without the production of undesirable physiologicaleffects such as nausea, dizziness, gastric upset and the like.Compositions for use according to the invention may comprise one or morecarriers, excipients and/or diluents as set out below.

According to one aspect of the invention there is provided a formulationcomprising one or more SS-14 analogs of the invention in a medicamentfor the treatment, prophylaxis or management of a disorder associatedwith neoplastic cells.

According to one embodiment of the invention there is provided a methodfor treating a human or non-human animal with a disorder associated withneoplastic cells comprising the step of administering a formulationcomprising an SS-14 analog of the invention. The disorder associatedwith neoplastic cells may be, for example, colorectal cancer, gastriccancer, prostate cancer, cancer in the pancreas.

Non-human animals which may be treated typically include mammals,particularly livestock and domestic animals such as dogs, cats, rabbits,guinea pigs, hamsters, mice, rats, horses, goats, sheep, pigs and cows.

Depending on the mode of administration, various forms of thecompositions may be used. Thus, pharmaceutical compositions may beformulated in conventional manner using readily available ingredients.The active ingredients comprising a peptide analog may be incorporated,optionally together with other active substances, with one or moreconventional carriers, diluents and/or excipients, to produceconventional preparations such as tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments, soft and hardgelatine capsules, suppositories, sterile injectable solutions, sterilepackaged powders, and the like.

Examples of suitable carriers, excipients and diluents, are lactose,dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calciumphosphate, alginates, tragacant, gelatine, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, watersyrup, water, water/ethanol, water/glycol, water/polyethylene glycol,propylene glycol, methyl cellulose, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate, mineral oil or fattysubstances such as hard fat or suitable mixtures thereof. Thecompositions may additionally include lubricating agents, wettingagents, emulsifying agents, suspending agents, preserving agents,sweetening agents, flavoring agents, and the like. The compositions ofthe invention may be formulated so as to provide quick, sustained ordelayed release of the active ingredient after administration to thepatient by employing procedures well known in the art. Compositions maybe in an appropriate dosage form, for example, as an emulsion or inliposomes, niosomes, microspheres, nanoparticles or the like.

Administration of compositions for use in the invention may take placeby, any of the conventional routes, e.g. by inhalation, orally, rectallyor parenterally, such as by intramuscular, subcutaneous, intraarticular,intracranial, intradermal, intraocular, intraperitoneal, intrathecal,intravenous injection although this depends on the condition to betreated. The injection may even be performed directly into an affectedsite (for example, by stereotaxic injection). Local administration mayalso be performed, e.g. at an affected site e.g. by use of a catheter orsyringe. Treatment by topical application of a composition, e.g. anointment, to the skin is also possible for appropriate conditions.Optionally administration may be performed at intervals, e.g. 2 or moreapplications, e.g. 2-4 applications at hourly, daily, weekly or monthlyintervals, e.g. several times a day, or every 3-5 days, or atfortnightly, monthly or quarterly intervals.

The SS-14 analogs of the invention are present in the compositions fromabout 0.01% to about 99% by weight of the formulation, typically fromabout 0.1 to about 50%, for example 10%. The compositions may beformulated in a unit dosage form, e.g. with each dosage containing fromabout 0.01 mg to about 1 g of the active ingredient, e.g. 0.05 mg to 0.5g, for a human, e.g. 1-100 mg. The precise dosage of the active compoundto be administered and the length of the course of treatment will, ofcourse, depend on a number of factors including for example, the age andweight of the subject, the specific condition requiring treatment andits severity, and the route of administration. Generally however, aneffective dose may lie in the range of from about 10 μg/kg body weightto about 60 μg/kg body weight of an SS-14 analog of the invention,typically about 10 μg/kg to about 20 μg/kg per day, depending on thesubject to be treated and the dosage form, taken as a single dose. Thusfor example, an appropriate daily dose for an adult may be from 0.5 mgto 2 g per day, e.g. 1.0 to 500 mg of an SS-14 analog of the inventionper day.

The peptides of the invention can be made using any number of techniquesknown in the art. The peptides may be synthesized by a suitable method,such as by exclusively solid-phase techniques, by partial solid-phasetechniques, by fragment condensation, or by classical solution addition.For example, the techniques of exclusively solid-state synthesis are setforth in numerous textbooks including, for example, “Solid-Phase PeptideSynthesis”, Stewart and Young, Freeman & Co., San Francisco, 1969. Thefragment condensation method of synthesis is exemplified in U.S. Pat.No. 3,972,859 (Aug. 3, 1976). Other available syntheses are exemplifiedby U.S. Pat. No. 3,842,067 (Oct. 15, 1974) and U.S. Pat. No. 3,862,925(Jan. 28, 1975).

Common to coupling-type syntheses is the protection of the labile sidechain groups of the various amino acid moieties with suitable protectinggroups which will prevent a chemical reaction from occurring at thatsite until the group is ultimately removed. Usually also common is theprotection of an alpha-amino group on an amino acid or a fragment whilethat entity reacts at the carboxyl group, followed by the selectiveremoval of the alpha-amino protecting group to allow subsequent reactionto take place at that location. Accordingly, it is common that, as astep in the synthesis, an intermediate compound is produced whichincludes each of the amino acid residues located in its desired sequencein the peptide chain with various of these residues linked to theside-action protecting groups.

Typical protecting groups, coupling agents, reagents and solvents suchas, but not limited to those, listed below have the followingabbreviations as used herein and in the claims. One skilled in the artwould understand that the compounds listed within each group may be usedinterchangeably. Further, one skill in the art would know other possibleprotecting groups, coupling agents and reagents/solvents; these areintended to be within the scope of this invention.

Abbreviated Designation Protecting Groups

-   -   Ada Adamantane acetyl    -   Alloc Allyloxycarbonyl    -   Allyl Allyl ester    -   Boc tert-butyloxycarbonyl    -   Bzl Benzyl    -   Fmoc Fluorenylmethyloxycarbonyl    -   OBzl Benzyl ester    -   OEt Ethyl ester    -   OMe Methyl ester    -   Tos (Tosyl) p-Toluenesulfonyl    -   Trt Triphenylmethyl    -   Z Benzyloxycarbonyl

Abbreviated Designation Coupling Agents

-   -   BOP Benzotriazol-1-yloxytris-(dimethyl-amino) phosphonium        hexafluorophosphate    -   DIC Diisopropylcarbodiimide    -   HBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate    -   PyBrOP Bromotripyrrolidinophosphonium hexafluorophosphate    -   PyBOP Benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium        hexafluorophosphate    -   TBTU        O-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N′,N′-tetramethyluronium        tetrafluoroborate Reagents

Abbreviated Designation and Solvents

-   -   ACN Acetonitrile    -   AcOH Acetic acid    -   Ac₂O Acetic acid anhydride    -   AdacOH Adamantane acetic acid    -   Alloc-Cl Allyloxycarbonyl chloride    -   Boc₂O Di-tert butyl dicarbonate    -   DMA Dimethylacetamide    -   DMF N,N-dimethylformamide    -   DIEA Diisopropylethylamine    -   Et₃N Triethylamine    -   EtOAc Ethyl acetate    -   FmocOSu 9-fluorenylmethyloxy carbonyl N-hydroxysuccinimide ester    -   HOBT 1-Hydroxybenzotriazole    -   HF Hydrofluoric acid    -   MeOH Methanol    -   Mes (Mesyl) Methanesulfonyl    -   NMP 1-methyl-2-pyrrolidinone    -   nin. Ninhydrin    -   i-PrOH Iso-propanol    -   Pip Piperidine    -   PP 4-pyrrolidinopyridine    -   Pyr Pyridine    -   SRIF Somatotropin release inhibiting factor    -   SST Somatostatin receptor    -   TEA Triethylamine    -   TFA Trifluoroacetic acid    -   THF Tetrahydrofuran    -   Triflate (Trf) Trifluoromethanesulfonyl    -   Trf₂O Trifluoromethanesulfonic acid Anhydride

The compounds herein described may have asymmetric centers. All chiral,diastereomeric, and racemic forms are included in the present invention.Many geometric isomers of olefins and the like can also be present inthe compounds described herein, and all such stable isomers arecontemplated in the present invention.

Several octapeptides were synthesized by solid-phase techniques, using aRink-amide MBHA resin as the solid support. Generally, the linearpeptide sequences are assembled via the Fmoc-strategy, and employingHBTU/HOBt/DIEA or PyBOP/HOBt/DIEA as coupling agents. Cyclization iscarried with excess iodine in DMF. The peptides are then cleaved fromthe resin, with the simultaneous deprotection of side chains, using acleavage mixture composed of TFA, water, and anisole. The peptides canbe purified by any conventional technique including, for example,RP-HPLC and characterization by analytical HPLC and MALDI-FTMS.

The SS-14 analogs of the invention can be synthesized by classicalsolution synthesis, but are typically synthesized by solid-phasetechnique. A chloromethylated resin or a hydroxymethylated resin can beused. For example, these peptides having a free carboxyl C-terminus aretypically synthesized as taught in U.S. Pat. No. 4,816,438 issued Mar.28, 1989, the disclosure of which is incorporated herein by reference.Solid-phase synthesis is conducted in a manner to stepwise add aminoacids in the chain beginning at the C-terminus. Side-chain protectinggroups, which are well known in the art, are included as a part of anyamino acid that has a particularly reactive side chain, and optionallymay be used in the case of others such as Trp, when such amino acids arecoupled onto the chain being built upon the resin. Such synthesisprovides the fully protected intermediate peptidoresin. Typically a Rinkamide based resin will be used. Rink Amide AM resin and Rink Amide MBHAresin are the most popular resins for the synthesis of peptide amide byFmoc chemistry. Coupling of the first Fmoc amino acid to the resin canbe achieved using the same protocol as for the solid phase peptidesynthesis. Detachment of peptide amides from these supports can beaffected in a single step by treatment with 95% TFA and appropriatescavengers. Thus, somatostatin analogs of the invention can bechemically synthesized in vitro. Furthermore, the SS-14 analogs of thisinvention can be synthesized wherein a radiolabel-binding moiety iscovalently linked to the peptide during chemical synthesis in vitro,using techniques well known to those with skill in the art. Suchpeptides covalently-linked to the radiolabel-binding group duringsynthesis are advantageous because specific sites of covalent linkageare defined.

The invention has been described above, the following specificembodiments are provided to further illustrate the invention. Thespecific examples below are not meant to limit the scope of theinvention.

EXAMPLES Synthesis of the Peptides

The following peptides were synthesized (modified residues are shown inbold type):

-   1. (4-Amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂-   2. (4-Amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂-   3. (4-Amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Thr-NH₂-   4. (4-Amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Asp-NH₂-   5. (4-Amino)-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂-   6. (4-Amino-3-iodo)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂-   7. (4-Amino-3-iodo)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂-   8.    (4-Amino-3-iodo)-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂-   9.    (4-Amino-3-iodo)-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂-   10. D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂

The following notations were used: “(4-amino)-D-Phe” denotes(4-aminophenyl)-D-phenylalanine, i.e.2-amino-3-(4-aminophenyl)-propanoic acid, “(3-iodo)-Tyr” denotes(4-hydroxy-3-iodophenyl)-L-tyrosine, i.e.2-amino-3-(4-hydroxy-3-iodophenyl)-propanoic acid, and“(4-amino-3-iodo)-D-Phe” denotes (4-amino-3-iodophenyl)-D-phenylalanine,i.e. 2-amino-3-(4-amino-3-iodophenyl)-propanoic acid.

The following protected amino acids were used: Fmoc-Thr(tBu)-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Boc-D-Phe-OH, available fromCalbiochem-Novabiochem, a division of Merck, Darmstadt, Germany. Some ofthe reagents (coupling agents HBTU and DIC, H-(3-iodo)-Tyr-OH,Boc-(4-amino)-D-Phe-OH) were purchased from Chem-Impex International,Wood Dale, Ill., except Boc-(4-amino-Fmoc)-D-Phe-OH, which was obtainedfrom Bachem, Torrance, Calif. The DMF was purchased from FisherScientific and treated with sodium aluminosilicate molecular sieves (4 Ånominal pore diameter) obtained from Sigma and Amberlite IR120 (plus)cation exchange resin, strongly acidic. Methylene chloride (DCM) wasdistilled from calcium hydride. Rink amide MBHA resin and PyBOP werepurchased from Calbiochem-Novabiochem, (Merck, Darmstadt, Germany). Thereactions were monitored by thin-layer chromatography (TLC) carried outon EM Science Merck silica gel coated on aluminum plates (0.2 mmthickness, 60 F₂₅₄) using UV light (254 nm) as the visualizing agent and10% ninhydrin in ethanol, bromocresol green in ethanol or 7% ethanolicphosphomolybdic acid and heat as developing agents. Silica gel 60(230-400 mesh) purchased from EM Science was used for columnchromatography.

The Kaiser test was used as a qualitative assay for the presence orabsence of free amino groups during reactions on solid phase.

The peptides were constructed by manual solid phase synthesis methods,using Rink amide MBHA resin with a substitution level of 0.54 mmol/g.The activation/coupling agents used were PyBOP:HOBt:DIEA, 4:4:8 (eq), orHBTU:HOBt:DIEA, 4:4:8 (eq). All couplings and deprotections were carriedout in DMF. Generally, to couple the first amino acid, 5 equivalentswere used and the coupling reaction completed overnight. Following this,4 equivalents of the commercially available protected amino acids wereused, and coupling reaction times were 4 to 8 hours, allowing fordifferences in reactivity among amino acids. For coupling of cysteine,an activation/coupling method was chosen that did not involve use ofbase, namely DIC:HOBt, 1:1 (mmol), in a mixture of DCM/DMF 1/1 (vol), tominimize epimerization. Reaction time was about to 2 h.

The resin was initially swollen in DCM for at least 30 min. Deprotectionof the Fmoc group from the resin and throughout the synthesis wasachieved with 20% piperidine in DMF. After deprotection, thepeptide-resin was washed 2 times with DMF for 1 minute, then 2 times DCM(1 min.), then again with DMF (2×1 min). After coupling, four washeswith DMF were followed by washing two times with DCM, then two timeswith MeOH, two times with DCM, and again four times with DMF. Thewashing process was sometimes repeated.

Cyclization was accomplished using excess I₂ (10 eq) in DMF (20 mL) for3 h. The peptide-resin was then washed 10 times with DMF, or more, untilthe solvent coming out of the reaction vessel was clear. To removetraces of iodine completely, a THF wash was followed by a 0.5 M Na₂S₂O₂rinse, followed by DCM, MeOH, DCM, THF and DMF. Finally, thepeptide-resin was rinsed twice with DCM and dried in the dessicatorunder high vacuum to completely remove any trace of iodine bysublimation.

For final deprotection and simultaneous cleavage of the final peptidefrom the resin, a cocktail composed of 9.5 mL TFA, 0.25 mL H₂O and 0.25mL anisole was used. Water and anisole were appropriate scavengers forremoving t-butyl cations, which could otherwise attack the aromaticresidues. The “cleavage cocktail” and the peptide-resin were separatelycooled in ice, then the solution was added over the resin, and thevessel was shaken for exactly one hour at room temperature. The liquidphase was removed by filtration, and the resin washed 5 times with neatTFA. The collected filtrate containing the target peptide as a TFA saltwas taken to dryness and traces of TFA were removed by azeotropicdistillation with toluene, three times. The crude peptide was cooled inan ice bath, and cold ether added. A white precipitate formed, which waswashed with ether, isolated by centrifugation, dissolved in water, andthe aqueous solution lyophilized overnight. All the crude peptidesshowed one major peak by HPLC.

Synthesis of the Amino Acid Building Blocks

Boc-(4-amino)-D-Phe-OH is commercially available.

The peptidomimetic amino acid H-(3-iodo)-tyrosine (10) was commerciallyavailable. It was converted to Fmoc-(3-iodo)-tyrosine (11, Scheme 1).The reaction was carried out in the presence of 10% NaHCO₂, overnight.

Fmoc-(3-Iodo)-tyrosine (11) To H-(3-iodo)-Tyr-OH (1.00 g, 3.26 mmol)were added 7 mL DMF and 10 mL acetone, and the suspension was cooled to0° C. in an ice bath. After 10 min, 10% NaHCO₃ (w/v) (7 mL, 8.15 mmol)was added dropwise, followed by FmocOSu (1.32 g, 3.91 mmol). Another 25mL of DMF were added, and the milky suspension was stirred vigorously atroom temperature, overnight. (Note: the starting material was insolublein water, DMF, THF, dioxane, acetonitrile, acetone, CHCl₃ and DCM). Thesolvent was removed under reduced pressure and 25 mL of water wereadded. The water was extracted once with EtOAc (5 mL) to removeimpurities insoluble in water. The aqueous layer was cooled in an icebath and acidified to pH approx. 1 with 2N NaHSO₄ and extracted fivetimes with EtOAc (5×5 mL). The EtOAc layer was then washed twice withNaCl sat., dried over Na₂SO₄, filtered and concentrated to result in aclear oil. This was dried overnight on a vacuum manifold to become awhite foamy solid. Chloroform (50 mL) was added, and a white precipitateformed. The precipitate (product) was filtered on a Buchner funnel, anddried overnight to yield 1.41 g (82%).

¹H NMR (DMF, 400 MHz): δ(ppm) 7.87 (d, J=8 Hz, 2H), 7.63 (t, J=8 Hz,2H), 7.57 (s, 1H), 7.39 (m, 2H), 7.33 (m, 2H), 7.06 (d, J=8 Hz, 1H),6.77 (d, J=8 Hz, 1H), 4.18 (m, 3H), 4.02 (m, 1H), 2.94 (dd, J₁=16 Hz,J₂=4 Hz, 1H), 2.72 (dd, J₁=16 Hz, J₂=12 Hz, 1H)

MS: ESI (m/z): calculated for C₂₄H₂₀NO₅I [MH]⁺ expected 530. Found 530,[M+Na]⁺ expected 552. Found 552, [M−H]⁻ expected 528. Found 528

[α]_(D) ²⁵=+9.04° (c=0.88, MeOH)

TLC(CHCl₃:MeOH:AcOH 90:10:1, bromocresol green), R_(f)=0.33.

To prepare the protected monoiodinated amino acidBoc-(4-amino-3-iodo-)-D-Phe-OH (12, Scheme 2) a simple procedure wasperformed, utilizing a “classical” iodination reagent iodine chlorideICl.

Boc-(4-amino-3-iodo)-D-Phe-OH (12) To a 100 mL round-bottomed flask wereadded Boc-(4-amino)-D-Phe-OH (300 mg, 1.070 mmol) and 5 mL glacialacetic acid. The flask was covered in aluminum foil. While stirring, ICl(174 mg, 1.070 mmol) was added dropwise, and the flask was immediatelycapped. The reaction mixture was stirred for 10 min. An excess of 0.5 NNa₂S₂O₃ was added to stop the reaction and quench remaining unreactediodine (the solution turned from brown to slightly yellow). Ethylacetate and water were added and the layers separated. The aqueous layerwas back-extracted twice with ethyl acetate. The combined organic layerwas further washed with 0.5 N Na₂S₂O₃ (3×5 mL) and saturated NaCl (3×5mL), dried over Na₂SO₄ and filtered. The filtrate was concentrated andthe residue dried on a vacuum manifold. The crude was purified by columnchromatography using as eluent EtOAc:hexanes:AcOH 5:5:0.1 (mL) to yield118.4 mg of product (27%). A portion of the product was further purifiedby HPLC and lyophilized to provide a sample (yellowish solid) foranalytical purposes.

¹H NMR (DMSO, 400 MHz): δ 7.46 (s, 1H), 7.03 (d, J=8.0 Hz, 2H), 6.99 (d,J=8 Hz, 1H), 6.73 (d, J=8 Hz, 1H), 3.96 (m, 1H), 2.83 (dd, J₁=16 Hz,J₂=4 Hz, 1H), 2.63 (dd, J₁=16 Hz, J₂=12 Hz, 1H), 1.33 (s, 9H)

¹³C NMR (DMSO, 400 MHz): δ 173.4, 155.1, 146.6, 138.5, 129.6, 127.3,113.9, 82.9 (C-I), 77.9, 55.4, 34.9, 28.2

MS: ESI (m/z) calculated for C₁₄H₁₉N₂O₄I [MH]⁺ expected 407. Found 407,[M+Na]⁺ expected 429. Found 429, [M−H]⁻ expected 405. Found 405;MALDI-FTMS: [M+Na]⁺ expected 429.0282. Found 429.0290

m p. 68-80° C. (with decomposition)

[α]_(D) ²⁵=−27.5° (c=1, MeOH) (starting material [α]_(D) ²⁵=−26.9°, c=1,MeOH)

TLC (EtOAc:hexanes:AcOH 5:5:0.1 mL, ninhydrin) R_(f)=0.36

HPLC analytical: 1 mg/mL sample concentration, 10 μL injection, 10-90%B, (A: H₂O with 0.1% TFA v/v; B: AcCN with 0.1% TFA v/v) at 1 mL/minover 30 min, A=220 nm, R_(t)=18.81 min. The starting material in thesame conditions exhibited an R_(t)=12.16 min.

The Boc protecting group survived the reaction conditions. Thediiodinated Boc-(4-amino-3,5-diiodo)-D-Phe-OH was obtained as a minorproduct (the ratio of monoiodinated:diiodinated was about 2:1 in oneinstance of carrying out the reaction). This mixture was separated bycolumn chromatography (50% yield of monoiodinated compound). Thebuilding block was then incorporated into the peptide structures with anunprotected aromatic amine, since this amine is much less reactive foramide bond formation reactions.

Synthesis of the Peptides

The peptide amides were synthesized by solid phase peptide synthesis(SPPS), following Fmoc protocols. The Rink amide MBHA resin was used asthe solid support. Cleavage from this resin can be accomplished by asingle step treatment with 95% TFA, providing amides in high yields andpurities.

Structure of the Rink Amide MBHA Resin

The following protected amino acids were used: Fmoc-Thr(tBu)-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH,Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Boc-D-Phe-OH,Boc-(4-amino-Fmoc)-D-Phe-OH, and the unusual iodinated amino acids,Fmoc-(3-iodo)-Tyr-OH and Boc-(4-amino-3-iodo)-D-Phe-OH, prepared asdescribed herein.

Coupling reagents used included PyBOP or HBTU in combination with HOBtand DIEA. Disulfide bridges were formed by reaction with excess iodinein DMF while the side chain protected peptides were on the resin. Inthis case, iodine functioned to remove the Acm protecting groups andoxidize the free —SH groups to form the disulfide bond.

A few illustrative examples are presented in Schemes 3, 4, 5 and 6, anddetailed procedures are presented herein.

The synthesis of peptide 2 is detailed in Scheme 3. The Fmoc-protectedresin was swollen in DCM and the Fmoc group deprotected using 20%piperidine/DMF. The first amino acid, Fmoc-Asp(OtBu)-OH was coupledusing a preactivated (2 min) mixture of amino acid with HBTU, HOBt andDIEA (4:4:2:8 eq relative to theoretical resin loading of 0.54 mmol/g).The remaining amino acids were added using Fmoc protocols. The lastamino acid, Boc-(4-amino-Fmoc)-D-Phe-OH was introduced using DIC:HOBt(1:1 mmol) in DMF. The linear, protected peptide was then cyclized onthe resin, with excess iodine (5 eq) for 3 h. The side chain Fmoc wasremoved with 20% piperidine in DMF. The cyclic peptide was cleaved fromthe resin, with the concomitant removal of the remaining protectinggroups Boc and t-butyl, by treatment with TFA:H₂O:anisole (95:2.5:2.5%).The peptide was purified by RP-HPLC.

Scheme 4 illustrates the synthesis of peptide 5. After removal of theexisting Fmoc protecting group from the resin, the first amino acid,Fmoc-Thr(tBu)-OH (4 eq) was coupled to the resin using PyBOP/HOBt/DIEAin DMF, (4:2:8 eq to theoretical maximal loading). Fmoc-(3-I)-Tyr-OH wascoupled overnight using the same coupling mixture. The last amino acid,Boc-(4-amino-Fmoc)-D-Phe-OH, was coupled as above, using DIC/HOBt. Thecyclization was effected with iodine. The remaining steps were carriedout as above.

Scheme 5 illustrates the synthesis of peptide 6. The first amino acid,Fmoc-Thr(tBu)-OH was coupled as presented above. The last amino acid,Boc-(4-amino-3-iodo)-D-Phe-OH (12), was coupled to the peptide chainusing DIC:HOBt (1:1 eq) in DMF, for 10 h. The peptide was cyclized withiodine, thoroughly washed, and cleaved from the resin with concomitantremoval of the protecting groups.

Scheme 6 illustrates the synthesis of peptide 8. The first amino acid,Fmoc-Thr(tBu)-OH was coupled as presented above. The Fmoc-(3-I)-Tyr-OHwas also introduced as above. The last amino acid,Boc-(4-amino-3-iodo)-D-Phe-OH (12), prepared with the side chain aminefree, was coupled to the peptide chain using DIC:HOBt (1:1 eq) in DMF,for 9 h. The peptide was cyclized with iodine, thoroughly washed, andcleaved from the resin with concomitant removal of the protectinggroups.

(4-Amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (1) was synthesizedby manual SPPS, on Rink amide resin (s.l. 0.54 mmol/g), 0.2 mmol scale,in 228 mg crude yield, as noted above. A portion of the product waspurified using 25% B and UV detection at λ 280 nm, to yield 16.9 mg purepeptide. Analytical conditions were: gradient 10-90% B, R_(t)=14.33 minand isocratic 26% B, R_(t)=11.13 min. MALDI-FTMS (m/z): [MH]⁺ calcd. forC₅₀H₆₈N₁₂O₁₀S₂ expected 1061.4695. Found 1061.4733.

(4-Amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (2) was synthesizedby manual SPPS, on Rink amide MBHA resin (0.54 mmol/g), 0.37 g, 0.2 mmolscale, as noted above. The crude yield was 143.8 mg (67%). A portion ofthe peptide (40 mg) was purified with 25% β isocratic, and UV detectionat λ 280 nm to yield 13.3 mg pure peptide. Analytical conditions were:gradient 10-90% B over 30 min, R_(t)=17.12 min and isocratic 26% B,R_(t)=8.72 min. MALDI-FTMS (m/z): [MH]⁺ calcd. for C₅₀H₆₆N₁₂O₁₁S₂expected 1075.4488. Found 1075.4448.

(4-Amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Thr-NH₂ (3) wassynthesized as noted above, on 0.1 mmol scale, and 115 mg crude wasobtained. Purification of a portion of material was carried out usingisocratic conditions of 23% B and UV detection at λ 220 nm and 5 mg pureproduct were obtained. Analytical conditions: gradient 10-90% B over 30min, R_(t)=13.58 min and isocratic 20% B, R_(t)=11.59 min. MALDI-FTMS(m/z): [MH]⁺ calcd. for C₅₀H₆₈N₁₂O₁₀S₂ expected 1061.4695. Found1061.4650.

(4-Amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Asp-NH₂ (4) wassynthesized manually, as noted above, on a Rink amide MBHA resin (0.54mmol/g), 0.19 g, 0.1 mmol scale and 88.9 mg was obtained (82%). Aportion of the peptide was purified using isocratic conditions (20% B)and UV detection at λ 220 nm and 6.16 mg pure peptide was obtained.Analytical HPLC conditions: gradient 10-90% B over 30 min, R_(t)=13.59min, and isocratic 20%, R_(t)=11.26 min. MALDI-FTMS (m/z): [MH]⁺ calcd.for C₅₀H₆₆N₁₂O₁₁S₂ expected 1075.4488. Found 1075.4492.

(4-Amino)-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (5) wassynthesized by manual SPPS, 0.2 mmol scale, 0.37 g resin.Fmoc-(3-I)-Tyr-OH (11) (0.26 g, 0.5 mmol, 2.5 eq), PyBOP (0.26 g, 0.5mmol, 2.5 eq), HOBt (0.07 g, 0.5 mmol, 2.5 eq) and DIEA (0.17 mL, 1.0mmol, 5 eq) were mixed in DMF (20 mL) and added over the deprotectedpeptide-resin. The resulting mixture was opalescent because the aminoacid was not completely soluble. In spite of this problem, the couplingof this building block was successful. The resin was moved from thereaction vessel into a scintillation vial, and coupling carried outovernight. The resin was removed from the scintillation vial, washed ona Buchner funnel, several times, with DMF and DCM, then transferred backto the reaction vessel for subsequent couplings. The Kaiser test wasused to assess the completion of the reaction and the resin was washedagain with DMF (4×20 mL) and DCM (2×20 mL) and DMF (4×20 mL). Thebuilding of the peptide chain was resumed as usual. The last amino acidadded was Boc-(4-amino-Fmoc)-D-Phe-OH (0.25 g, 0.5 mmol, 2.5 eq)preactivated in the presence of PyBOP (0.42 g, 0.5 mmol, 2.5 eq), HOBt(0.12 g, 0.5 mmol, 2.5 eq) and DIEA (0.21 mL, 0.5 mmol, 5 eq), in 20 mLDMF. This residue was coupled for 18 h. For cyclization, only 5 eq ofiodine (0.25 g, 1.0 mmol) was used, for 3 h, in order to preventiodination of the aromatic residues. The peptide-resin was thenextensively washed as described in the general procedures and notessection, and dried for 2 h in a dessicator under high vacuum toeliminate the smallest trace of iodine. The side chain Fmoc protectinggroup was removed using 20% piperidine in DMF, for 1 h. The peptideresin was washed thoroughly and dried in a dessicator overnight inpreparation for final deprotection/cleavage from the resin. Finaltreatment with TFA/H₂O/anisole (9.5/2.5/2.5 mL), and usual work-upresulted in 237.7 mg crude peptide (quantitative). A portion of 23 mg ofthe crude product was purified using isocratic conditions, 25% B, and UVdetection at λ 210 and 220 nm, to yield 8.1 mg pure product. Analyticalconditions: gradient 10-90% B, 30 min, R_(t)=18.04 min, isocratic 26% B,R_(t)=16.45 min. MALDI-FTMS (m/z): [MH]⁺ calcd. for C₅₀H₆₂IN₁₂O₁₀S₂expected 1187.3662. Found 1187.378, [M+Na]⁺ expected 1209.3481. Found1209.3533.

(4-Amino-3-iodo)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (6) wasbuilt as noted above, on 0.2 mmol scale, but only 90 mg peptide-resinwere used to couple the last amino acid. The coupling mixture containedBoc-(4-amino-3-iodo)-D-Phe-OH (12) (0.044 g, 0.11 mmol, 1.5 eq), DIC(0.017 mL, 0.11 mmol, 1.5 eq) and HOBt (0.017 g, 0.11 mmol, 1.5 eq) in10 mL DMF and the reaction was allowed to proceed for 10 h. Cyclizationwas carried out with iodine (0.16 g, 2.0 mmol, 10 eq) for 3 h, and thepeptide-resin carefully washed and dried as illustrated above for 5. Thefinal deprotection/cleavage procedure was accomplished in 1 h, and afterlyophilization, 30.6 mg (53%) of crude peptide resulted. Purificationwas accomplished with 24% B in isocratic mode. The yield was 9.9 mg (13%based on theoretical loading level of the resin). Analytical HPLC:gradient 10-90% B, UV detection at λ 220 nm, R_(t)=18.9 min, isocratic25% B, R_(t)=9.32 min. MALDI-FTMS (m/z): [MH]⁺ calcd. forC₅₀H₆₂IN₁₂O₁₂S₂ expected 1187.3662. Found 1187.3689, [M+Na]⁺ expected1209.3481. Found 1209.3470.

(4-Amino-3-iodo)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (7) wassynthesized in the same manner as compound 6. A portion of 90 mgpeptide-resin was used for coupling of the final building block, whichresulted in 19.4 mg (37%) crude peptide. The peptide was purified with24% B and UV detection at λ 220 nm. The yield was 4.1 mg (7%).Analytical HPLC: gradient 10-90% B, 30 min, R_(t)=18.74 min andisocratic 22% B R_(t)=10.65 min. MALDI-FTMS (m/z): [MH]⁺ calcd. forC₅₀H₆₅IN₁₂O₁₁S₂ expected 1201.3454. Found 1201.3542, [M+Na]⁺ expected1223.3274. Found 1223.3296.

(4-Amino-3-iodo)-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (8)was synthesized as above, using 90 mg peptide resin. The couplings ofthe unusual building blocks were carried out as described above. Thecrude yield was 41 mg (64%). Purification was carried out with 25-50% Bover 30 min and UV detection at λ 220 nm. The yield was 4.1 mg (7.4%).Analytical conditions: gradient 10-90% B, 30 min, R_(t)=20.4 min andisocratic 25% R_(t)=16.77 min. MALDI-FTMS (m/z): [MH] calcd. forC₅₀H₆₆I₂N₁₂O₁₀S₂ expected 1313.2628. Found 1313.2644, [M+Na]⁺ expected1335.2448. Found 1335.2471.

(4-Amino-3-iodo)-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (9)was synthesized by manual SPPS as described above and 38.7 mg (60%)crude was obtained. Purification: 25-40% B, 30 min, UV detection at λ220 nm. The yield was 5 mg (7.8%). Analytical: gradient 10-90% B over 40min, R_(t)=17.62 min, isocratic 25% B R_(t)=10.41 min. MALDI-FTMS (m/z):[MH]⁺ calcd. for C₅₀H₆₄I₂N₁₂O₁₁S₂ expected 1327.2421. Found 1327.2423,[M+Na]⁺ expected 1349.224. Found 1349.2274.

The target molecules were purified and analyzed by RP-HPLC using VydacProtein Peptide C₁₈ columns. Column dimensions were 4.5×250 mm (90 Åsilica, 5 μm) for analytical and 22×250 mm (90 Å silica, 10 μm) forpreparative HPLC. The UV absorbance was monitored at 220 nm (280 nm insome cases). A binary system of water (A) and acetonitrile (B), bothcontaining 0.1% TFA, was used throughout. Preparative HPLC was carriedout at 10 mL/min flow rate, on two different instruments. One instrumentwas a system composed of two Waters 510 pumps and a dual wavelength UVabsorbance detector. The other instrument, which was also used foranalytical purposes as described below, was a Waters Millenium 2010system, composed of two Waters 510 pumps, a 715 Ultra WISP sampleinjector, and a 996 photodiode array detector (PDA), operated by aNecStar PC compatible computer. Two analytical HPLC profiles of the purecompounds were obtained on the Waters Millenium PDA system, using alinear gradient of usually 10-90% acetonitrile (B) in water (A) over 30min and an isocratic elution at 1 mL/min flow rate.

NMR spectra were obtained on a Varian HG-400 (400 MHz) and a Bruker AMX500 (500 MHz) spectrometers. Chemical shifts (δ) are reported in partsper million (ppm) relative to residual undeuterated solvent as aninternal reference. The following abbreviations were used to explainmultiplicities: s=singlet, d=doublet, t=triplet, q=quartet, dd=doubletof doublets, m=multiplet, b=broad.

Optical rotations were recorded on a Perkin-Elmer 241 polarimeter.

IR spectra were recorded on a Nicolet-Magna-IR 550 Series IIspectrometer. Mass spectroscopic analyses (ESI, MALDI-FTMS) were carriedout by the Scripps Center for Mass Spectrometry at The Scripps ResearchInstitute, La Jolla, Calif.

Conformational Analysis-NMR and Molecular Modeling Studies. Thestructures of all the compounds were confirmed by 1D and 2D ¹H NMR inDMSO at 500 MHz.

NMR Spectroscopy. The samples were dissolved in DMSO-d₆. The NMR spectrawere acquired on a Bruker AMX 500 spectrometer operating at 500 MHz andprocessed using FELIX2000 (Biosym/MSI, Inc.).

The ¹H NMR data are presented in Tables 3 and 4 (chemical shifts),Tables 5 and 6 (relevant backbone NOEs) and Tables 7 and 8 (couplingconstants J_(NH—C) ^(α) _(H), calculated φ angles, coupling constantsJ_(C) ^(α) _(H—C) ^(β) _(Hl)/J_(C) ^(α) _(H—C) ^(β) _(Hh), calculatedside-chain populations and temperature coefficients). The followingnotations are observed: C^(α)H and H_(α) are used interchangeably todenote the proton linked to the α C atom. H_(α) ³ denotes the protonlinked to the α C atom of residue 3. The subscripts l and h denote low-and high-field resonances, respectively.

TABLE 3 The Chemical Shifts of Compounds 1, 3, 5, 8, 10 Analog 1 3 5 810 D-Phe¹NH 8.05 7.95 7.95 7.98 7.97 Hα 4.19 4.05 4.01 4.09 4.06 Hβ3.21/2.93 3.06/2.72 3.02/2.77 3.05/2.70 3.06/2.69 H2 7.34 7.03 7.03 7.6 7.62 H3 7.28 6.56 6.52 / / H4 7.34-7.28 / / / / H5 7.28 6.56 7.03 6.7 6.7  H6 7.34 7.03 6.52 7.09 7.06 Cys²NH 9.12 9.12 9.07 9.12 9.14 Hα 5.245.24 5.25 5.25 5.23 Hβ 2.78/2.78 2.80/2.80 2.84/2.84 2.83/2.83 2.80/2.80Tyr³NH 8.58 8.56 8.65 8.57 8.61 Hα 4.61 4.59 4.61 4.65 4.6  Hβ 2.80/2.652.79/2.64 2.82/2.66 2.82/2.67 2.78/2.64 H2 6.9  6.89 6.9  6.9  7.49 H36.63 6.6  6.62 6.62 / H5 6.63 6.6  6.62 6.62 6.77 H6 6.9  6.89 6.9  6.9 6.98 OH 9.17 9.2  9.2  9.2  10.1  D-Trp⁴NH 8.66 8.64 8.67 8.65 8.73 Hα4.22 4.19 4.23 4.24 4.26 Hβ 2.99/2.76 2.97/2.73 2.99/2.76 3.00/2.773.00/2.77 H1 10.75  10.7  10.78  10.7  10.8  H2 7.07 7.04 7.07 7.08 7.1 H4 7.44 7.41 7.44 7.43 7.44 H5 6.98 6.96 6.98 6.98 6.98 H6 7.05 7.067.05 7.06 7.05 H7 7.31 7.3  7.31 7.31 7.31 Lys⁵NH 8.35 8.34 8.34 8.368.35 Hα 3.92 3.88 3.95 3.9  3.91 Hβ 1.71/1.24 1.67/1.22 1.72/1.211.70/1.25 1.67/1.18 Hγ 0.69 0.67 0.66 0.69 0.62 Hδ 1.27 1.26 1.24 1.271.25 Hε 2.5  2.5  2.5  2.54 2.48 NH₂ 7.56 7.59 7.63 7.59 7.56 Val⁶NH7.54 7.52 7.54 7.54 7.48 Hα 4.31 4.3  4.39 4.32 4.31 Hβ 2.07 2.04 2.082.06 2.05 CH₃γ 0.91/0.87 0.89/0.86 0.88/0.86 0.90/0.87 0.90/0.87 Cys⁷NH8.55 8.54 8.59 8.55 8.56 Hα 5.03 5.03 5.09 5.03 5.03 Hβ 2.80/2.802.80/2.80 2.79/2.79 2.81/2.81 2.80/2.80 X-Asp⁸NH 8.1  8.11 8.16 8.1 8.1  Hα 4.56 4.56 4.54 4.57 4.57 Hβ 2.64/2.64 2.64/2.64 2.62/2.622.82/2.66 2.65/2.65 NH₂ 7.54 7.58/7.36 7.58/7.37 7.59/7.34 7.57/7.34

TABLE 4 The Chemical Shifts of Compounds 2, 4, 6, 7, 9 Analog 2 4 6 7 9D-Phe¹NH 7.9  7.94 7.93 7.92 7.92 Hα 4.06 4.08 4.04 4.03 4.04 Hβ3.16/2.81 3.06/2.78 3.14/2.79 3.12/2.75 3.11/2.74 H2 7.13 7   7.12 7.657.63 H3 6.67 6.54 6.7  / / H4 / / / / / H5 6.67 6.54 6.7  6.7  6.7  H67.13 7   7.12 7.1  7.12 Cys²NH 9.27 9.15 9.29 9.25 9.27 Hα 5.44 5.3 5.45 5.45 5.46 Hβ 2.79/2.79 2.79/2.79 2.77/2.77 2.83/2.83 2.81/2.81Tyr³NH 8.67 8.67 8.7  8.69 8.71 Hα 4.6  4.61 4.57 4.6  4.59 Hβ 2.79/2.672.81/2.65 2.75/2.63 2.80/2.68 2.79/2.66 H2 6.91 6.89 7.43 6.91 7.47 H36.62 6.62 / 6.63 / H5 6.62 6.62 6.77 6.63 6.78 H6 6.91 6.89 6.96 6.916.99 OH 9.22 9.2  10.1  9.1  10.1  D-Trp⁴NH 8.7  8.67 8.77 8.72 8.79 Hα4.23 4.22 4.24 4.23 4.25 Hβ 2.98/2.72 2.99/2.76 2.98/2.70 3.00/2.743.02/2.73 H1 10.8  10.81  10.7  10.7  10.7  H2 7.06 7.07 7.08 7.07 7.09H4 7.41 7.43 7.41 7.41 7.41 H5 6.96 6.97 6.97 6.98 6.98 H6 7.04 7.047.04 7.06 7.05 H7 7.3  7.3  7.29 7.31 7.33 Lys⁵NH 8.37 8.37 8.38 8.388.37 Hα 3.94 3.92 3.95 3.94 3.93 Hβ 1.74/1.25 1.69/1.22 1.70/1.171.73/1.22 1.69/1.18 Hγ 0.66 0.67 0.6  0.66 0.59 Hδ 1.26 1.26 1.26 1.261.23 Hε 2.55 2.5  2.52 2.52 2.5  NH₂ 7.67 7.68 7.6  7.59 7.58 Val⁶NH7.48 7.56 7.47 7.52 7.46 Hα 4.43 4.36 4.45 4.42 4.43 Hβ 2.04 2.08 2.012.03 2.01 CH₃γ 0.88/0.84 0.91/0.87 0.88/0.87 0.88/0.86 0.89/0.86 Cys⁷NH8.63 8.63 8.64 8.64 8.66 Hα 5.3  5.28 5.31 5.31 5.3  Hβ 2.79/2.792.94/2.84 2.75/2.75 2.82/2.82 2.81/2.81 X-Thr⁸NH 8.19 8.01 8.2  8.188.18 Hα 4.28 4.21 4.29 4.27 4.28 Hβ 4.04 4.03 4.03 4.03 4.02 CH₃γ 1.041.05 1.05 1.04 1.03 OH 5.2  5.38 5.2  5.23 5.18 NH₂ 7.63/7.37 7.50/7.287.67/7.37 7.62/7.34 7.64/7.34

TABLE 5 Observed Backbone NOEs^(a) for Compounds 1, 2, 3, 4 and 5 NOE 12 3 4 5 H_(α) ¹-NH² s s s s s H_(α) ²-NH² m m m m H_(α) ²-NH³ s s m sH_(α) ²-H_(α) ⁷ m m m m H_(α) ³-NH³ H_(α) ³-NH⁴ H_(α) ⁴-NH⁴ m m m m mH_(α) ⁴-NH⁵ s s s s s H_(α) ⁵-NH⁵ m m m m m H_(α) ⁵-NH⁶ m m m m mNH⁵-NH⁶ m m m m m H_(α) ⁶-NH⁶ m m m m m H_(α) ⁶-NH⁷ s s s s s NH⁶-NH³ mm m m H_(α) ⁷-NH⁷ m m m m H_(α) ⁷-NH⁸ s s s s s NH⁷-NH⁸ m w m H_(α)⁸-NH⁸ m m m m ^(a)The NOEs corresponding to distances ≦2.5 Å areclassified as strong (s); the NOEs corresponding to distances >2.5 and≦3.5 Å are classified as medium (m); the NOEs corresponding todistances >3.5 and ≦4.5 Å are classified as weak (w).

TABLE 6 Observed Backbone NOEs for Compounds 6, 7, 8, 9 and 10 NOE 6 7 89 10 H_(α) ¹-NH² s s s s H_(α) ²-NH² m m m s m H_(α) ²-NH³ s s s s sH_(α) ²-H_(α) ⁷ m m m m m H_(α) ³-NH³ m H_(α) ³-NH⁴ s H_(α) ⁴-NH⁴ m m mm m H_(α) ⁴-NH⁵ s s s s s H_(α) ⁵-NH⁵ m m m m m H_(α) ⁵-NH⁶ m m m mNH⁵-NH⁶ m m m m m H_(α) ⁶-NH⁶ m m m m m H_(α) ⁶-NH⁷ s s s s s NH⁶-NH³ mm w m m H_(α) ⁷-NH⁷ m m m m H_(α) ⁷-NH⁸ s s s s s NH⁷-NH⁸ m m H_(α)⁸-NH⁸ m m m m m ^(a)The NOEs corresponding to distances ≦2.5 Å areclassified as strong (s); the NOEs corresponding to distances >2.5 and≦3.5 Å are classified as medium (m); the NOEs corresponding todistances >3.5 and ≦4.5 Å are classified as weak (w).

TABLE 7 Coupling Constants J_(NH-C) _(I) _(H), Calculated φ values^(a),J_(C) _(I) _(H-C) _(θ) _(H) Coupling Constants, Side ChainPopulations^(a) and Temperature Coefficients (−ppb/K) of the AmideProtons^(b) in Compounds 1, 2-5. Side-chain Calculated population−Δppm/K Residue J_(NH-C) _(α) _(H) φ angles J_(αβl)/J_(αβh) g⁻, t, g⁺ NH1 D-Phe¹ / / 5.70/7.84 0.23, 0.54, / 0.23 Cys² / / / / 6.8 Tyr³ 8.22−146, −94 9.44/6.50 0.35, 0.47 4.3 0.18 D-Trp⁴ 4.38 171, 69, 9.05/6.780.20, 0.44, 6.4 −102, −18 0.36 Lys⁵ 8.22 −146, −94 ~14, 1, f(t) ~ f(g⁺)4 too small Val⁶ 9.64 −131, −109 8.81 0.57 0.9 Cys⁷ 9.32 −136, −104 / /5.5 Asp⁸ 7.12 −154, −86, / / 3.3 2 79, 40 4-amino- / / 5.68/8.66 0.30,0.49 / D-Phe¹ 0.21 Cys² 9.33 −136, −104 / / 7.7 Tyr³ 8.71 −142, −978.83/7.25 0.51, 0.36, 3.5 0.13 D-Trp⁴ 6 −90, −30, 8.52/6.78 0.20, 0.486.5 79, 161 0.31 Lys⁵ 7.26 −153, −87, 4.41/11.0 0.77, 0.21, 4.3 78, 420.07 Va1⁶ 8.87 −140, −99 6.21 0.32 0.6 Cys⁷ 9.25 −136, −103 / / 5.8 Thr⁸8.24 −146, −94 5.19 0.24 5.3 3 4-amino- / / 6.08/8.59 0.27, 0.49, D-Phe¹0.21 Cys² 9.49 −133, −106 / / 7.1 Tyr³ 8.63 −142, −97 6.73/8.78 / 4.3D-Trp⁴ 4.56 −101, −19, 7.00/8.23 0.22, 0.45, 6.5 70, 170 0.33 Lys⁵ 7.67−150, −90, 4.89/10.54 0.72, 0.21, 4.2 72, 48 0.07 Va1⁶ 9.54 −132, −1074.45 0.17 0.2 Cys⁷ 9.34 −135, −104 / 5.5 Asp⁸ 8.06 −147, −93 / 4.3 44-amino- / / 5.58/8.70 0.30, 0.20, / D-Phe¹ 0.50 Cys² 8.86 −141, −99 / /6.8 Tyr³ 8 −148, −92 7.85/6.10 0.42, 0.25, 4.7 0.33 D-Trp⁴ 6.44 −86,−34, 9.26/6.38 0.17, 0.55, 7.15 82, 158 0.27 Lys⁵ 8.4 −145, −95 ~14, 1,f(t) ~ f(g⁺) 3.9 too small Val⁶ 9.01 −139, −101 7.14 0.41 0.8 Cys⁷ 9.47−134, 106 4.62/10.43 0.71, 0.18, 5.9 0.10 D-Thr⁸ 8.6 97, 143 4.8 0.2 5.35 D-Phe¹- / / 6.12/918 0.20, 0.25, / NH₂ 0.55 Cys² 8.69 −142, −98 / /6.5 Tyr³ 7.74 −150, −90, 7.85/6.02 overlap 3.7 70, 50 D-Trp⁴ 4.15 −104,−16, 8.16/7.14 0.20, 0.49, 5.3 173, 67 0.32 Lys⁵ 8.12 −147, −93 ~14, 1,f(t) ~ f(g⁺) 3.8 too small Va1⁶ 9.82 −128, −112 8.17 0.35 0.5 Cys⁷ 9.62−132, −108 / / 5 D-Asp⁸ 7.9 −66, −54, / overlap 3.22 91, 148 ^(a)See NMRspectroscopy experimental section. ^(b)In DMSO solution, amide protonsthat have temperature coefficients (−Λppb/K) in the range of 0-2.0−ppb/K are considered to be involved in an intramolecular hydrogenbonding or are solvent shielded. Values greater than 4.0 −ppb/K areconsidered to be completely solvent exposed.

TABLE 8 Coupling Constants J_(NH-C) _(I) _(H), Calculated φ values^(a),the Coupling Constants J_(C) _(I) _(HC) _(θ) _(H), the Side ChainPopulations^(a) and Temperature Coefficients (−ppb/K) of the AmideProtons^(b) for Compounds 6-10 Side-chain −Δppm/ Calculated population KResidue J_(NH-C) _(α) _(H) φ angles J_(αβl)/J_(αβh) g⁻, t, g⁺ NH 63-iodo-4- / / 6.20/8.78 0.23, 0.51, / amino-D-Phe¹ 0.26 Cys² 9.12 −138,−102 / / 7.4 3-iodo-Tyr³ 8.03 −148, −92 8.13/6.21 0.26, 0.45, 4 0.30D-Trp⁴ 5.11 −97, −23, 8.94/7.45 0.10, 0.52, 5.6 73, 167 0.38 Lys⁵ 8.77−141, −98 4.62/10.3 0.70, 0.20, 4 0.11 Val⁶ 9.98 −122, −117 6.95 0.4 1.8Cys⁷ 9.48 −134, −106 / / 6 Thr⁸ 8.6 −143, −97 4.39 0.16 5 7 3-iodo-4- // 6.43/9.19 0.32, 0.47, / amino-D-Phe¹ 0.21 Cys² 9.34 −136, −104 / / 5.8Tyr³ 8.54 −143, −96 7.90/6.59 0.32, 0.39, 4.3 0.29 D-Trp⁴ 6.24 −88, −32,7.88/6.23 0.32, 0.42, 6.3 80, 160 0.26 Lys⁵ 8.56 −143, −97 ~14, 1, f(t)~ f(g⁺) 4 too small Val⁶ 9.31 −136, −104 6.19 0.33 0.5 Cys⁷ 9.01 −139,−101 / / 5.5 Thr⁸ 8.22 −146, −94 4.7 0.19 3.6 8 3-iodo-4- / / 5.35/8.550.34, 0.48, / amino-D-Phe1 0.17 Cys2 8.91 −140, −100 / / 5.8 Tyr3 8.1−147, −93 8.07/8.58 0.43, 0.50, 4.3 0.07 D-Trp4 4.46 171, 69, 8.56/6.810.20, 0.49, 6.3 −102, −18 0.32 Lys5 8.37 −145, −95 ~14, 1, f(t) ~ f(g+)4 too small Val6 9.12 −138, −102 6.33 0.34 0.5 Cys7 9.28 −136, −104 / /5.5 Asp8 8.27 −146, −94 6.85/6.44 0.35, 0.39, 3.6 0.26 9 3-iodo-4- / /6.28/8.98 0.37, 0.44, / amino-D-Phel 0.19 Cys2 9.12 −138, −102 / / 73-iodo-Tyr3 7.9 −149, −91, 7.91/6.88 0.31, 0.40, 4 66, 53 0.29 D-Trp44.86 −99, −21, 8.94/7.22 0.12, 0.52, 5.8 72, 168 0.35 Lys5 8.51 −144,−96 ~14, 1, f(t) ~ f(g+) 4.1 too small Val6 9.53 −133, −107 8.15 0.5 0.6Cys7 8.12 −138, −102 / / 5.7 Thr8 8.51 −144, −96 4.95 0.21 5.1 103-iodo-4- / / 5.70/8.70 0.21, 0.56, / amino-D-Phel 0.23 Cys2 9.49 −134,−106 / / 5.7 3-iodo-Tyr3 7.81 −149, −91, 8.62/4.64 / 3.9 −69, 51 D-Trp45.02 167, 73, 8.70/6.30 0.23, 0.50, 5.8 −97, −23 0.27 Lys5 8.37 −145,−95 ~14, 1, f(t) ~ f(g+) 4 too small Val6 9.4 −135, −105 8.55 0.54 0.1Cys7 8.93 −140, −100 / / 5.4 Asp8 8.37 −145, −95 / / 3.6 ^(a,b)as inTable 7

Tables 7 and 8 show the χ₁ rotamer populations computed on the basis ofthe measured J_(C) ^(α) _(H—C) ^(β) _(H) coupling constants using thethree-state rotamer model. The side chain conformations which arerelevant for somatostatin-like binding activity are those of D-Phe¹,D-Trp⁴, and Lys⁵. For the residues in position 1, in compounds 1, 2, 3,6, 7, 8, 9 and 10 the most populated χ_(|) rotamer is consistently thetrans. This conformation allows the aromatic side chain of the D-Phe¹ tobe located in the vicinity of the bridging disulfide region. There aresome minor differences between these analogs regarding the orientationof the D-Phe¹ aromatic side chain relative to the disulfide bridge. Inmost analogs this side-chain adopts a trans orientation, which resultsin close spatial proximity of the disulfide bridge and the aromaticring. This orientation has also been reported for Sandostatin®. In theanalogs with D configuration at C^(α) in position 8, the side chainprefers a g⁺ orientation, which brings the aromatic ring of position 1far from the disulfide bridge.

As for the side chains of D-Trp⁴ and Lys⁵, the χ₁ rotamers in thebioactive compounds are trans and g⁻, respectively, as shown in Tables 7and 8. These rotamers allow a close proximity between the side chains ofD-Trp⁴ and Lys⁵, which is confirmed by the upfield shift observed forthe C^(γ)H resonances of Lys⁵, caused by the D-Trp⁴ aromatic ringcurrent. These conformational preferences are very similar to thosefound for the parent compound Sandostatin®.

These studies demonstrate that all analogs adopt conformations verysimilar to each other. Furthermore, the conformations of these compoundsare very similar to those adopted by Sandostatin® and its activeanalogs. The backbone conformation can be described as an antiparallelβ-sheet containing a type II′ β-turn with D-Trp in the i+1 position, andmost structures are folded about Phe³ and Val⁶.

In the molecules containing iodine, there seems to be a preference forthe aromatic side chains to situate themselves close to one another.This tendency may create a large hydrophobic area, which may bind to aspecific hydrophobic zone on the receptors. Alternatively, the presenceof the large, hydrophobic iodine may induce a conformation that favorsbinding to receptors hsst2 and hsst5, but not hsst3.

The conformational analysis data show that the analogs studied havegreat similarity among each other and compared to Sandostatin®. Mediumstrength NOEs between the NH protons of Val⁶ and Lys⁵ and the absence ofNOEs between the NH protons of Lys⁵ and D-Trp⁴ suggest a type II′ β-turnwith D-Trp in the i+1 position for these compounds. This is consistentwith the low temperature coefficients of the Val⁶NH protons and withstrong sequential NOEs between Lys⁵NH and D-Trp⁴H_(α), medium strengthNOEs between Val⁶NH and Lys⁵H_(α), and medium strength NOEs betweenLys⁵NH and Lys⁵H_(α).

The β-sheet-like conformation found for the other parts of the backboneis consistent with the J_(NH—C) ^(α) _(H) coupling constants and withthe high temperature coefficients measured for the NH⁴ and NH⁷resonances (Tables 7 and 8). These temperature coefficients are in fullagreement with the solvent exposure expected for NH⁴ and the NH⁷ protonsin the antiparallel β-sheet structure. The β-sheet-like conformation isalso supported by the strong sequential C^(α)H—NH NOEs (see the H_(α)²—NH³, the H_(α) ³—NH⁴, the H_(α) ⁶—NH⁷ and the H_(α) ⁷—NH⁸ NOEs, and bythe H_(α) ²—H_(α) ⁷ NOE (Tables 5 and 6). The H_(α) ²—H_(α) ⁷ NOE alsoindicates that the disulfide bridge is a good mimetic of a β-VI turn forwhich this type of H_(α) to H_(α) NOE is typically observed.

The conformational analysis of Sandostatin® suggested that this moleculeadopts multiple conformations such as a β-sheet structure andconformations which contain a helical fold in the C-terminal portion.All the analogs studied showed the NH⁶—NH³ NOEs consistent with aβ-sheet structure, but only the analogs with Asp in position 8 showedthe NH⁷—NH⁸NOEs, characteristic for a partial helical fold in thisregion. These NOEs indicate that the partially helical structures aremore populated in the compounds containing an Asp residue, than in thecompounds with a Thr residue in position 8.

NMR spectroscopy. The resonance assignments were carried out using ¹HNMR, TOCSY (total correlation spectroscopy) and ROESY (rotating framenuclear Overhauser) experiments. The TOCSY experiment is used to assignthe spin systems and the ROESY experiment allows the sequentialassignments of the amino acid spin systems from observed sequential NOEconnectivities d_(C) ^(α) _(H—HN) or d_(HN—HN) or possibly d_(C) ^(β)_(H—HN).

The J_(NH—C) ^(α) _(H) and J_(C) ^(α) _(H—C) ^(β) _(H) couplingconstants were obtained from 1D spectra and from sections ofcross-peaks, obtained from the DQF-COSY spectra. Measured NH—C^(α)H andC^(α)H—C^(β)H coupling constants allowed us to estimate the ranges of φand χ₁ torsion angles. The vicinal coupling constant J_(NH—C) ^(α) _(H)is related to torsion angle φ (which represents the rotational statearound skeletal single bond NH—C^(α)H) by a Karplus-type equation:J _(NH—C) ^(α) _(H) =A cos²|φ±60°|−B cos|φ±60°|+C

where:

(+) is for the D configuration and (−) is for the L configuration,respectively and coefficients: A=8.6, B=1.0 and C=0.4.

The values of coupling constants J_(NC—C) ^(I) _(H) and thecorresponding estimated (1) angle values are listed in Tables 7 and 8.

The fraction of the individual conformers f₁ (x₁) may be estimated byusing the following conventional expressions:

(a) for the L residue

f(g⁻)=(J_(HI—Hθ1)−J_(G))/(J_(T)−J_(G)) for the gauche (−) conformation

f(t)=(J_(HI—Hθ2)−J_(G))/(J_(T)−J_(G)) for the trans conformation

f(g⁺)=1−[f(g⁻)+f(t)] for the gauche (+) conformation (b) and for the Dresidue:

f(t)=(J_(HI—Hθ2)−J_(G))/(J_(T)−J_(G)) for the trans conformation.

f(g⁺)=(J_(HI—Hθ1)−J_(G))/(J_(T)−J_(G)) for the gauche (+) conformation

f(g⁻)=1−[(f(g⁻)+f(t)] for the gauche (−) conformation.

The values J_(T)=13.56 Hz and J_(G)=2.60 Hz for trans and gauchecoupling were used to calculate the rotameric populations of nonaromatic residues, while the values J_(T)=13.85 Hz and J_(G)=3.55 Hzwere used for the aromatic residues.

Different intensities of the amide to β proton NOEs and the α to βproton NOEs, from ROESY spectra, allow the assignment of thediastereotopic β protons. Once the diastereotopic methylene β protonsare assigned, the rotameric population f(t), f(g⁺), f(g⁻) can becalculated using the previous equations. However, in some cases becauseof the overlapping of the β protons or the absence of significant NOEs,the diastereotopic assignment cannot be performed. In such cases it isnot possible to distinguish between the two possible rotamericpopulations.

Molecular modeling. The computer simulations were carried out on an SGIIRIX 6.5 workstation and Challenge computer (Silicon Graphics). TheInsight II and DISCOVER programs were used for the molecular mechanics,dynamics calculations and visualizations.

Initially, 200 structures were generated using DGII/InsightII, followedby a restrained minimization with a CVFF91 force field and theNewton-Ralphson VA09A algorithm with a convergence criterion of 0.001Kcal/molÅ. All the calculations were carried out in vacuo and a distancedependent dielectric constant was used to take into account the solventeffects. In the simulations, the peptide bonds were maintained in thetrans conformation.

The torsion angles, the NOE distances and hydrogen-bonding patterns ofthese structures were compared with the values derived from NMRmeasurements. A Karplus-type equation was used to compute the torsionangles consistent with the measured J_(NH—C) ^(α) _(H) couplingconstants, and an error of ±30° was tolerated. In the case of selectionsbased on the hydrogen bonds, structures were retained in which NHprotons with a temperature coefficient greater than 2.0-ppb/K wereconsidered to be involved in a hydrogen bond. Structures not consistentwith these experimental constraints were discarded. A cut-off of 10Kcal/mol above the lowest energy conformer was used to sort outunrealistically high-energy conformations of the remaining structures.

A cluster analysis was performed by first extracting the lowest energyconformer out of all the conformations under investigation. Theseconformers served as a “seed” to grow a cluster as long as the selectedtorsion was within a 30° interval. The analysis then iterativelysearched for the next high-energy cluster until the highest family wasfound. Prior to every molecular dynamics simulation, the system wasequilibrated with 3 ps initialization dynamics. In attempts to carry outa thorough search of the accessible conformational space, the lowestenergy conformation of the clusters was subjected to 500 ps ofrestrained molecular dynamics at 1000 K with a step size of 1 fs. Theconformers that were consistent with experimental data were subjected tothe same cluster analysis as described above. Finally, the lowest energystructure of each cluster was chosen as the preferred conformation insolution of the somatostatin analogs.

Unrestrained molecular dynamics simulations at 300 K with adistance-dependent dielectric constant of 1 were carried out toinvestigate the equilibrium between these preferred conformations. Theselected conformers were first submitted to 3 ps of equilibrium,followed by a step size of 1 fs for a 5 ns simulation. Structures werecollected every 10 ps. During this process, conformational interchangeswere observed.

Biological Activity

The binding assays can be executed on membranes from CC531 cells (hsst2)and CHO-K1 cells (hsst1, 3 and 5), transfected with individual humansomatostatin receptor subtypes. The functional assays-inhibition ofgrowth hormone and prolactin secretion—cab be carried out with dispersedrat pituitary cells.

Female Wistar rats (Harlan, The Netherlands), weighing 180-200 g, arekept in an artificially illuminated room (08.30-20.30 h) with food andwater ad libitum. The animals are killed between 09.00 and 10.00 h bydecapitation. The pituitary glands are removed within 5 minutes afterkilling, the neuro-intermediate lobe is discarded and the anterior lobesare collected in calcium, magnesium-Hank's balanced salt solution (HBSS)supplemented with 1% fetal calf serum (FCS), penicillin (100 U/mL),streptomycin (100 μg/mL), fungizone (0.5 μg/mL) and sodium carbonate(0.4 μg/L final concentration).

Female anterior pituitary cells are isolated with dispase (see, e.g.,Oosterom et al., J Endocrinol. 100(3):353-60, 1984). The dispersed cellsare seeded at a density of 0.5−1×10⁵ cells per well in multiwell plates.The culture medium is Eagle's Minimum Essential Medium with Earle'ssalts supplemented with a 1-fold excess of nonessential amino acids, 1mM sodium pyruvate, 2 mM L-glutamine, penicillin (100 U/mL),streptomycin (100 μg/mL), and fungizone (0.25 μg/mL) and 10% fetal calfserum (Invitrogen, Breda, the Netherlands). Media and supplements areobtained from Gibco Bio-Cult Europe (Invitrogen, Breda, theNetherlands). The cells are allowed to attach for at least 3 days.Thereafter, medium is changed and 4-hr incubations with or without asomatotropin release inhibiting factor (SRIF) analogs is provided and 10nM GH-releasing hormone (GHRH) in 1 mL complete culture medium isinitiated, using about four dishes for every treatment group. Theresults of each experiment are expressed as percentile change of hormonerelease compared with control untreated dishes. The concentration of ratGH and PRL is determined by means of a commercially available rat GH andPRL assay (Amersham, United Kingdom).

For expression of the somatostatin receptor subtype sst 2 and sst-1,3,5in rat colon carcinoma (CC531) cells and Chinese hamster ovaries(CHO)-K1 cells, respectively, human sst1, sst2, sst3 or sst5 cDNA inpBluescript (pBS) are excised from pBS and inserted into the Nhe-1/SalIcloning site of a retroviral expression vector pCi-neo. The selection ismade by the geneticine resistance gene (G418). This vector is used tostably transfect (using DOTAP) CC531 and CHO-K1 cells. Stablytransfected CC531 and CHO-K1 cells are selected and cultured in RPMI1640 medium or nutrient mixture F12 (HAM) medium supplemented withpenicillin (100 U/mL), streptomycin (100 μg/mL), fungizone (0.25 μg/mL)and 10% FCS+geneticine (0.5 mg/mL), respectively.

Competitive binding experiments are performed with membranes preparedfrom CC531 (sst2) and CHO-K1 (sst-1,3,5) cells expressing the respectivehuman somatostatin receptor subtype (see, e.g., Reubi, Life Sci.36(19):1829-36 1985). Membrane preparations (corresponding to about25-50 μg protein) of cultured cells are incubated in a total volume of100 μl at room temperature for 45 minutes with 40.000 cpm ¹²⁵I-labeled[Tyr¹¹]-Somatostatin-14 (2000 Ci/mmol), in the presence of increasingamounts of unlabeled analogs. At the end of the incubation time, 1 mlice-cold Hepes buffer (10 mM Hepes, 5 mM MgCl₂ and 0.02 g/literbacitracin, pH 7.6) is added and membrane-bound radioactivity isseparated from unbound by centrifugation during 2 min at 14,000 rpm inan Eppendorf microcentrifuge. The remaining pellet is washed in ice-coldHepes buffer, and the final pellet is counted in a γ-counter. Specificbinding is defined as the total amount of radioligand bound minus thatbound in the presence of 1 μM unlabeled analog.

For a further description of biological assays please see: Hofland andLamberts, Front Horm Res. 32:235-52, 2004; Hofland et al., J ClinEndocrinol Metab. 89(4):1577-85, 2004; Ferone et al., Am J PhysiolEndocrinol Metab. 283(5):E1056-66, 2002; and Hofland et al.,Endocrinology. 136(9):3698-706, 1995).

Such experiments investigate the properties of RC-121 and use thisinformation to improve the properties of potency and selectivity at thehsst2 and hsst5 receptors and resistance to enzymatic degradation. Suchwork has led to increased interest in the role of the residues atpositions 1 and 8 for controlling receptor selectivity throughinteraction with extracellular loop III (ECLIII) located betweentransmembrane helices VI and VII.

Thus, a series of analogs of RC-121 were synthesized as described aboveand characterized as described herein. For example, the biologicalresults for [D-Phe¹-Asp⁸] (3), will demonstrate a selectivity forreceptors hsst2 and hsst5.

Residue 8 of the analogs was maintained as Thr or Asp, both D- andL-isomers, and modified the D-Phe in position 1. Asp was chosen as amodification for residue 8. For modifications in position 1, theunnatural amino acid (4-aminomethyl)-phenylalanine, which has thecharacteristics of both hydrophobicity and basicity was prepared. It wasbelieved that a weakly basic amino acid residue with an aromatic sidechain in position 1, such as the amino acid building block(4-amino)-D-Phe-OH, may enhance the potencies of the analogs.Accordingly, residue 1, H-D-Phe-OH, was replaced with (4-amino)-D-Phe-OHin RC-121, D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂. The bindingaffinities and functional assay results for this group of targetstructures are assayed accordingly.

Compounds with (4-amino)-D-Phe-OH in position 1 were expected to exhibitpotent and selective binding at receptors hsst2 and hsst5. Compound[(4-amino)-D-Phe¹-Thr⁸] (2) is expected to be selective at the hsst2receptor by an order of magnitude compared to the hsst5 receptor. Suchcompounds can serve as inhibitor of GH and PRL release.

To study the binding affinities of the analogs, iodination assays can beperformed. Radioiodination is a useful tool for the determination oftissue distribution of somatostatin receptors and for delivering toxiclevels of radiation to somatostatin receptor-positive tumors, sinceseveral types of cancers express high densities of somatostatinreceptors. Iodine has been extensively used for radiolabeling orchemical modification of peptides and proteins. Radioiodinated analogsof hormones are used in binding assays for displacement studies(competitive binding assays).

The peptides of the invention have two aromatic residues, Tyr and(4-amino)-D-Phe-OH. The next generation of modifications focused oniodination of the residues with aromatic side chains. Usingmonoiodinated building blocks 11 and 12, several iodinatedpeptidomimetics were synthesized and characterized.

Compound [(4-amino)-D-Phe¹-(3-iodo)Tyr³-Thr⁸] (6), was synthesized withan iodine on tyrosine, and [(4-amino-3-iodo)-D-Phe¹-Thr⁸] (7) with aniodine on 4-amino-D-phenylalanine. In addition, the effect of adding twomonoiodinated residues in the molecules can also be examined. Thus,compounds [(4-amino-3-iodo)-D-Phe¹-(3-iodo)Tyr³-Thr⁸] (9) and(4-amino-3-iodo)-D-Phe¹-(3-iodo)Tyr³-Asp⁸] (10), were prepared, theformer with Thr in position 8, the latter with Asp in position 8. Theactivities of target molecules with or without iodine are compared andthe activity related to their conformations.

Peptides [(4-amino-3-iodo)-D-Phe¹-Thr⁸] (7) and[(4-amino-3-iodo)-D-Phe¹-(3-iodo)-Tyr³-Thr⁸] (9) are expected toexhibited potent and selective binding for hsst2 and 5 receptors, aswell as increased ability to inhibit both GH and PRL.

The somatostatin analogs of the invention are selective for receptorshsst2 and hsst5, with inhibitory activity on GH and PRL comparable tothat of somatostatin.

β-methylated analogs of Trp were synthesized enantioselectively andincorporated into the cyclic hexapeptide analog L-363,301. The bindingpotencies and selectivities of c[Pro-Phe-(2R,3S)-β-MeTrp-Lys-Thr-Phe]and c[Pro-Phe-(2R,3R)-β-MeTrp-Lys-Thr-Phe] at somatostatin receptorshsst1-5 were determined, to further examine the results. In thosestudies, the (2R,3S) analog was more potent than the parent peptide,while the (2R,3R) analog was much less active. Both compounds bound toreceptor hsst2 in nanomolar range. The analogs proved to be highlyselective for hsst2, in contrast with the standard compound L-363,301,which binds well to both receptors hsst2 and hsst5.

The peptide containing the (2R,3S)-β-MeTrp diastereomer possesses the“correct” combination of features that allow it to bind very well tohsst2. This finding has consequences for the future design ofhsst2-selective somatostatin analogs.

Statistical analysis. The statistical significance of the differencebetween mean values was determined using one-way analysis of variance(ANOVA). When significant overall effects were obtained by this method,comparisons were made using Newman-Keuls multiple comparison test. Dataare expressed as mean±sem. A P value less than 0.05 was consideredsignificant. Calculation of the IC₅₀ values for displacement of¹²⁵I-labeled [Tyr¹¹]-Somatostatin-14 was performed using the GraphPadPrism (San Diego, Calif.) computerized program.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method of visualizing malignant cells havingsomatostatin receptor-2 (SST2) and/or somatostatin receptor-5 (SST5)that bind a somatostatin analog in a subject comprising administering tothe subject a compound selected from any one of4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQ ID NO:1);4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQ ID NO:2);4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Asp-NH₂ (SEQ ID NO:3);4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Thr-NH₂ (SEQ ID NO:4);4-amino-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQ IDNO:5); 4-amino-3-iodo-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQ IDNO:6); 4-amino-3-iodo-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQ IDNO:7);4-amino-3-iodo-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQID NO:8);4-amino-3-iodo-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQID NO:9); and D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQ ID NO:10),wherein the compound is labeled with a detectable label, and visualizingmalignant cells that bind the compound.
 2. The method of claim 1,wherein the compound comprises a di- or polyiodinated aromaticmodification of a Tyr at position 3 of SEQ ID NOs:1-10.
 3. The method ofclaim 1, wherein a radioactive element is linked to the compound.
 4. Thecompound of claim 3, wherein the radioactive element is selected fromthe group consisting of scandium-47, copper-67, gallium-72, yttrium-90,iodine-125, iodine-131, samarium-153, gadolinium-159, dysprosium-165,holmium-166, ytterbium-175, lutetium-177, rhenium-186, rhenium-188,astatine-211 and bismuth-212.
 5. A method of treating a somatostatinreceptor-2 (SST2) and/or somatostatin receptor-5 (SST5) cellproliferative disorder in a subject comprising administering to thesubject an effective amount of a compound selected from any one of4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQ ID NO:1);4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQ ID NO:2);4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Asp-NH₂ (SEQ ID NO:3);4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Thr-NH₂ (SEQ ID NO:4);4-amino-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQ IDNO:5); 4-amino-3-iodo-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQ IDNO:6); 4-amino-3-iodo-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQ IDNO:7);4-amino-3-iodo-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQID NO:8);4-amino-3-iodo-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQID NO:9); and D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQ ID NO:10)and evaluating the subject after treatment with the compound.
 6. Amethod as in claim 5, wherein the cell proliferative disorder is atumor, an acromegaly, and/or a diabetes.
 7. The method of claim 5,wherein the compound comprises a di- or polyiodinated aromaticmodification of a Tyr at position 3 of SEQ ID NOs:1-10.
 8. The method ofclaim 5, wherein a radioactive element is linked to the compound.
 9. Thecompound of claim 8, wherein the radioactive element is selected fromthe group consisting of scandium-47, copper-67, gallium-72, yttrium-90,iodine-125, iodine-131, samarium-153, gadolinium-159, dysprosium-165,holmium-166, ytterbium-175, lutetium-177, rhenium-186, rhenium-188,astatine-211 and bismuth-212.
 10. A method of promoting somatostatinreceptor-2 (SST2) and/or somatostatin receptor-5 (SST5) receptoractivity in a mammal in need thereof comprising administering to saidmammal an effective amount of a compound selected from any one of4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQ ID NO:1);4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQ ID NO:2);4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Asp-NH₂ (SEQ ID NO:3);4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Thr-NH₂ (SEQ ID NO:4);4-amino-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQ IDNO:5); 4-amino-3-iodo-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQ IDNO:6); 4-amino-3-iodo-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQ IDNO:7);4-amino-3-iodo-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQID NO:8);4-amino-3-iodo-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQID NO:9); and D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQ ID NO:10)or a pharmaceutically acceptable salt thereof wherein the activity ofthe receptor is increased relative to the absence of the compound.
 11. Amethod of treating prolactin-secreting adenomas, restenosis, diabetesmellitus, hyperlipidemia, insulin insensitivity, Syndrome X, angiopathy,proliferative retinopathy, dawn phenomenon, nephropathy, gastric acidsecretion, peptic ulcers, enterocutaneous and pancreaticocutaneousfistula, irritable bowel syndrome, Dumping syndrome, watery diarrheasyndrome, AIDS-related diarrhea, chemotherapy-induced diarrhea, acute orchronic pancreatitis, gastrointestinal hormone-secreting tumors, cancer,hepatoma, angiogenesis, arthritis, chronic allograft rejection,angioplasty, graft vessel bleeding or gastrointestinal bleeding, in amammal in need thereof, which comprises administering to said mammal aneffective amount of a compound selected from any one of4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQ ID NO:1);4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQ ID NO:2);4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Asp-NH₂ (SEQ ID NO:3);4-amino-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-D-Thr-NH₂ (SEQ ID NO:4);4-amino-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQ IDNO:5); 4-amino-3-iodo-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQ IDNO:6); 4-amino-3-iodo-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQ IDNO:7);4-amino-3-iodo-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH₂ (SEQID NO:8);4-amino-3-iodo-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQID NO:9); and D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Asp-NH₂ (SEQ ID NO:10)or a pharmaceutically acceptable salt thereof and evaluating the subjectafter treatment with the compound.